WO2015099535A1

WO2015099535A1 - Thrombocidin-derived antimicrobial peptides

Info

Publication number: WO2015099535A1
Application number: PCT/NL2014/050909
Authority: WO
Inventors: Sebastianus Antonius Johannes Zaat; Paulus Hendricus Simon KWAKMAN; Jan Wouter Drijfhout
Original assignee: Academisch Ziekenhuis Leiden H.O.D.N. Lumc; Academisch Medisch Centrum
Priority date: 2013-12-24
Filing date: 2014-12-24
Publication date: 2015-07-02

Abstract

The invention relates to thrombocidin-derived antimicrobial peptides, pharmaceutical compositions comprising the peptides and to uses thereof for in the treatment or prevention of microbial, bacterial, fungal, viral and parasitic infection.

Description

Title: Thrombocidin- derived antimicrobial peptides

The invention relates to the field of biochemistry and medicine. More specifically the invention relates to the field of antimicrobial peptides and to counteracting bacterial, viral, fungal and parasitic infections, and controlling inflammation.

Antimicrobial peptides (AMPs) are components of the innate immune system of many organisms, including vertebrates, invertebrates and plants, and are part of the first line of defence against invading microorganisms. In humans, the most extensively studied antibacterial proteins are those in neutrophils and epithelial tissues. Blood platelets of humans and rabbits also contain antibacterial proteins which are released upon thrombin activation and likely are involved in defence against bloodstream infections such as infective endocarditis. AMPs offer protection from invading pathogens. They show potent antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, parasites and/or viruses. The smaller AMPs (usually about 15-40 amino acids) act largely by disrupting the structure or function of microbial cell membranes, they do not target single defined molecular structures. Therefore, as opposed to conventional antibiotics, they are effective regardless of the metabolic activity of bacteria. Human AMPs such as defensins and cathelicidin (LL-37) are present in leukocytes and secreted by various epithelia in skin and mucosal surfaces. In addition to their antimicrobial activity, AMPs are important effector molecules in inflammation, immune activation, and wound healing. AMPs are quite diverse in sequence and secondary structure, but share some common properties. They are usually cationic, amphipathic and exert their microbicidal effect by compromising the bacterial membrane integrity. Interaction of AMPs with the anionic membrane surface of the target microbes leads to membrane permeabilization, cell lysis and death. It is generally accepted that the cytoplasmic membrane is the main target of most AMPs, whereby accumulation of peptide in the membrane causes increased permeability and loss of barrier function resulting in leakage of cytoplasmic components and cell death. Conventional antibiotics kill bacteria by binding to targets such as an epitope on the cell wall, or targets in bacterial protein and DNA or RNA synthesis. Pathogenic bacteria develop resistance more rapidly by modifying the antibiotics targets so that antibiotics are no longer capable of binding these targets. A major advantage of AMPs over conventional antibiotics is that resistance does not readily develop. One reason for this is that they do not target single defined molecular structures (epitopes) like conventional antibiotics do, but act on the cell membrane killing microorganisms. AMPs can be particularly useful in counteracting so-called biofilms and their associated infections (BAI), which are surface -attached cellular agglomerates of microorganisms, mostly bacteria but also fungi. Biofilms contribute significantly to bacterial resistance to conventional antibiotics. Biofilms are associated with various pathological conditions in humans such as cystic fibrosis, colonization of inserted or indwelling medical devices and dental plaque formation, dental implant infection and wounds. Counteracting BAI with conventional antibiotics is further insufficient for a number of reasons including stimulation of the release of pro-inflammatory microbial compounds, insufficient penetration of the biofilm and inactivation or degradation in the blood as result of the necessary systemic administration. Other advantages of AMPs over

conventional antibiotics include the rapid onset of killing, the fact that they are biodegradable, which alleviates the current concern about residual antibiotics in the environment, and have concomitant anti-inflammatory activity.

Two major microbicidal proteins from human platelets are thrombocidin- 1 (TC-1) and thrombocidin-2 (TC-2), which are derivatives of CXC- chemokines CXCL7/NAP-2 and CTAP-III, respectively, differing from these chemokines by the absence of the two carboxyterminal amino acids. This C- terminal deletion is imperative for high microbicidal activity. Chemokines are primarily known for their role in inflammation where they attract and activate leukocytes. Based on their structure, chemokines are divided into four classes, depending on the number and spacing of the cysteines in their N-terminally part (C, CC, CXC and CX3C, in which X is any residue). The chemotactic activity of chemokines is mediated by the specific recognition of one or more chemokine receptors on target cells. The receptor recognition domain in CXC-chemokines such as the TC- 1 precursor CXCL7/NAP-2 involves a highly conserved Glu-Leu-Arg (ELR) motif located at the N-terminus of the proteins. Contrary to the domains of chemokines required for immunomodulatory activity, chemotaxis and receptor recognition, the localization of specific domains of chemokines required for their antimicrobial activity has not yet been characterized.

Various natural and synthetic antimicrobial peptides are used to develop novel antimicrobial agents, but currently no such agents have advanced to clinical application. The currently known AMPs still have a few drawbacks. An important disadvantage of known AMPs is that their activity is strongly affected in the presence of physiological salt concentrations and/or in complex biological matrices like plasma. Several mechanisms may be responsible for the significantly lower activity of AMPs in the presence of plasma, like inactivation of the peptide by plasma components, such as enzymatic degradation or non-availability due to nonspecific binding of the peptide to plasma components. AMPs that are resistant to plasma components are important as potential systemic therapeutic agents as well as for topical applications, for instance in treatment of infected wounds and medical implant related infection and inflammation. When applied systemically, AMPs might be bound by plasma components and/or proteolytically degraged within the circulation or in tissues. In addition, also wound fluids contain substantial protease activity, and implanted biomaterials are rapidly covered by plasma components from the hosts fluids. Thus, due to the sensitivity to plasma components, applicability of many AMPs will be limited, for instance to topical applications, but even for such applications the sensitivity to plasma components could affect the efficacy of AMPs.

There is thus a clear need for novel AMPs with high antimicrobial activity, preferably having activity in presence of physiological concentrations of NaCl like in PBS. AMPs that are resistant to plasma components are particularly needed, mainly as potential systemic therapeutic agents and/or therapeutic agent effective against biofilm infections associated with for instance medical devices such as implants. It is an object of the present invention to provide novel potent short thrombicidin- derived peptides with antimicrobial and anti-biofilm activity. It is a further object of the invention to provide such peptides that retain antimicrobial activity in biologically relevant media, such as in phosphate buffered saline and in the presence of plasma, e.g. human plasma. Novel peptides of the invention overcome shortcomings of conventional antibiotics and have improved properties over known antimicrobial peptides, in particular because they are highly potent and resistant to plasma. It is a further object of the invention to provide

antimicrobial peptides that have a particularly high antimicrobial activity against pathogenic microorganisms in biofilm- associated infections. Further preferred peptides of the invention have a low toxicity, e.g. a low hemolytic activity and/or anti-inflammatory (microbial compound-neutralizing) activity as evidenced by LPS and S. aureus neutralizing activity. The peptides of the invention exert potent, broad spectrum antimicrobial activities against a variety of microorganisms, have rapid antimicrobial activities and can be used as therapeutic or prophylactic agents.

The present inventors identified peptide regions within the sequence of TC-1 and TC-2 with microbicidal activity when synthesized as peptides and analysed the influence of changes in the composition of such peptides for their microbicidal activity and specificity. Microbicidal peptides representing two regions in the TCs were identified, one in the N-terminus and one in the C-terminus of the TCs. Peptides from the N-terminal region including the CXC-motif had the most potent microbicidal activity. The identified TC-pep tides represent most of the positive charge of the TC molecules and due to their localization in the folded TC molecule are part of an overall positively charged face of TCs (Kwakman, P. H., J. Krijgsveld, L. de Boer, L. T. Nguyen, L. Boszhard, J. Vreede, H. L. Dekker, D. Speijer, J. W. Drijfhout, A. A. te Velde, W. Crielaard, H. J. Vogel, C. M.

Vandenbroucke-Grauls, and S. A. Zaat. 2011. Native thrombocidin-1 and unfolded thrombocidin- 1 exert antimicrobial activity via distinct structural elements.

J.Biol. Chem. 286:43506-43514). The expectation of the present inventors was to identify peptides with highest microbicidal activity in the C-terminal region, for the following reasons; (i) C-terminal truncation of NAP-2 and CTAP-III was imperative for high microbicidal activity, (ii) in X ray and NMR studies of CXC-chemokines the C-terminal part has been shown to form an oc-helix, a structure considered to be responsible for the activity of antibacterial proteins, (iii) positively charged residues, thought to be important for the activity of microbicidal proteins, are clustered in the C-terminal region of TCs, (iv) a peptide derived from the C- terminal region of platelet factor 4 (PF-4), a CXC-chemokine with homology to CXCL7/NAP-2, has previously been shown to possess antibacterial activity, and (v) interferon- gamma-inducible chemokines with antimicrobial activity were shown to be equipped with strongly cationic tails suggested to be important for their antimicrobial activity. Several of the C-terminal peptides indeed were active, but surprisingly, a domain with higher microbicidal activity was identified around the CXC-motif in the N-terminal part of TC.

Peptides Al, E2, L3, R4, C5, localized in the N-terminal region of TC-1, are particularly potent and show activity against both bacteria and fungi. Peptide L3 was identified as the most active TC-derived antimicrobial peptide. Al, E2, L3, R4, C5 contain 2 cysteine residues which in full-length TCs are involved in disulfide bridge formation with cysteines at positions 31 and 47 (numbering of LC- 1, Figure 1). All variants of L3 lacking one positively charged amino acid (replaced by the neutral alanine: R4A, K9A and K17A, see table 2) retained antifungal activity, and the variants lacking such amino acid at position 4 and 9 in addition still displayed antibacterial activity, although activity was decreased. Variants with substitutions of individual neutral residues by the positively charged lysine (L3K, C5K, M6K, C7K, I8K, T10K, T11K, S12K, G13K, I14K, H15K or P16K, see table 2) yielded several peptides with microbicidal activity equalling that of full- length thrombocidins. The lysine substituted peptides at least retained the same level of activity as peptide L3 and all even had increased staphylocidal activity, and depending on the position that is substituted increased activity against one or more of the other micro-organisms tested.

It was further found that variants of L3 wherein the two central threonines are substituted by aromatic amino acids tryptophan, tyrosine or phenylalanine, have potent bactericidal activity in biologically relevant media, i.e in PBS and in the presence of plasma (see Table 3). Peptide TC19 was identified as the most active antimicrobial peptide having such substitution of the two central threonines with antimicrobial activity in PBS which is retained in the presence of 50% plasma. Replacement of individual amino acids in TC19, insertion of the aromatic amino acid tryptophan, threonine or phenylalanine between amino acids 9 (W) and 10 (S), N-terminal elongation with one or two of these aromatic amino acids and replacement of one or more amino acids with their corresponding D- amino acids resulted in peptides that retained or even improved antimicrobial activity (Tables 3, 4, 7 and 8). For instance, peptides TC82, TC85 and TC94 show a pattern of plasma stability similar to that of TC19, where TC82 and TC85 have slightly better bactericidal activity than TC19. Peptide TC84 even has superior activity, with very low LC99.9 values.

Further, peptides according to the invention have a low toxicity. As demonstrated in the Examples peptide TC19 shows less than 20% hemolysis in PBS in concentrations that are able in efficiently inhibit microorganisms (up to 51.2 μΜ) and even less than 5% hemolysis in the presence of plasma in

concentrations up to 204.8 μΜ.

Peptides of the invention are further demonstrated to have antiinflammatory properties, including microbial compound-neutralizing activity. As demonstrated in the Examples, peptide TC19 has LPS and S. aureus neutralizing activity, as evidenced by the ability to inhibit LPS-induced IL- 12P40 production and S. aureus induced IL-8 production by blood cells.

Hence, a peptide according to the present invention has high antimicrobial activity against micro-organisms and preferably is resistant against plasma, such as human plasma, has low toxicity and anti-inflammatory properties, making the peptide particularly suitable for not only topical, but also systemic administration and for use against biofilm associated infections, such as infection of medical devices and implants.

Accordingly, the present invention provides a synthetic, isolated or recombinant peptide of 10-25 amino acids comprising at least 10 amino acids of amino acid sequence LRCMCIKWWSGKHPK, or at least 10 amino acids of a variant of said sequence,

said peptide having antimicrobial, antibacterial, antiviral, antifungal, antiparasitic and/or anti-inflammatory activity; said variant sequence having one or more of the following modifications:

substitution of up to two amino acids selected from the group consisting of K and R by an amino acid selected from the group consisting of A, C, F, H, G, I, L, M, N, P, Q, S, T, V, W and Y;

substitution of up to four amino acids selected from the group consisting of L, C, M, I, W, T, S, G, H and P by an amino acid selected from the group consisting of K and R;

substitution of up to 5 amino acid selected from the group consisting of L, C, M, I, W, T, S, G, H and P by another amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W and Y;

substitution of one or more of K by R and/or R by K;

removal of one or both of C, or substitution of one or both of C by a negatively charged amino acid or a non-natural amino acid;

insertion or addition of one or two amino acid selected from the group consisting of A, C, F, H, G, I, L, M, N, P, Q, S, T, V, W and Y.

In amino acid sequences or variants thereof as defined herein amino acids are denoted by single-letter symbols. These single-letter symbols and three- letter symbols are well known to the person skilled in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (He) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine.

As used herein, a "negatively charged amino acid" refers to an amino acid that has a negative charge at physiological pH, i.e. at a pH of 7.3-7.4, preferably glutamic acid (E) and aspartic acid (D) and/or non-natural amino acids having a negative charge at physiological pH. Preferred negatively charged amino acids are glutamic acid (E) and aspartic acid (D).

A peptide of the invention has antimicrobial activity, preferably antibacterial, antifungal and/or antiviral activity, more preferably antibacterial and/or antifungal activity. Further, a peptide of the invention preferably has antimicrobial and anti-biofilm activity. Preferred peptides further have antiinflammatory activity. The term "antimicrobial activity" of a peptide as used herein refers to counteracting growth or proliferation of at least one microbe, e.g. a bacterium, a virus and/or a fungus, and includes inhibition, reduction or prevention of growth or proliferation as well as killing of the microbe. A microbe is an organism that is microscopic, i.e. usually too small to be seen by the naked human eye. Microbes are very diverse, they include bacteria, viruses, fungi, archaea, protozoans and microscopic algae. Similarly, the term "antibacterial activity", "antiviral activity", "antifungal activity" and "antiparasitic activity" as used herein refers to counteracting growth or proliferation of, respectively, a bacterium, a virus, a fungus and a parasite, in general and includes inhibition, reduction or prevention of growth or proliferation as well as killing thereof. Antimicrobial activity is for instance expressed as the inhibitory concentration (IC) or lethal concentration (LC). The ICx or LCx as used herein refer to the lowest peptide concentration which kills at least x% of microbes after 2 hours. For instance, LC99.9 refers to the lowest peptide concentration which kills >99.9% of cells of the microbial strain of the species under investigation. Antimicrobial, antibacterial, antiviral, antifungal and antiparasitic activity can be measured by methods known in the art. Two of such methods are detailed in the Examples of this application and involves an in vitro liquid assay for determination of antibacterial and antifungal activity. In this method microbes, e.g. bacteria or fungi, are incubated, for instance for 2 hours, with different concentrations of a peptide according to the invention, e.g. with serial 2-fold dilutions thereof. Subsequently, the microbe-peptide mixture is incubated at 37°C (e.g. for bacteria) or 30°C (e.g. for C. neoformans) in or on a suitable culture medium, such as blood agar plates, to establish the number of surviving and/or killed microbes as compared to a sample of microbes which have not been incubated with peptide but which has further been processed in the same way, for instance the next day or after 48 hours. Another method detailed in the Examples of this application peptides were incubated with lxlO⁶ CFU/ml of a mid- logarithmic suspension of S. aureus strain JAR060131 in phosphate buffered saline (PBS; pH 7.4), without or with addition of a pooled plasma at a final concentration of 50%. Antimicrobial activity is expressed as the 99.9% lethal concentration (LC99.9), i.e., the lowest peptide concentration at which 99.9% of bacteria were killed after 2 hours of incubation at 37°C under shaking conditions.

The term "anti-biofilm activity" of a peptide as used herein refers to counteracting growth or proliferation of at least one microbe, e.g. a bacterium, a virus and/or a fungus, in biofilm associated infections and includes inhibition, reduction or prevention of growth or proliferation as well as killing of the microbe. For determination of anti-biofilm activity, a suitable method is described in the Examples. This method involves determination of the IC50 after incubation of a peptide for 24 hours at 37°C with lxlO⁸ CFU/ml of S. aureus strain JAR060131 in biofilm-adjusted BM2 in 96-wells polypropylene plates, optionally coated with plasma by overnight incubation with 20% plasma at 4°C, removal of planktonic bacteria by four washes with PBS and staining of biofilms with crystal violet. After solubilization with ethanol, the optical density at 590 nm is determined as a measure of biofilm mass.

Virus plaque assays may be used to assess the antiviral activity of a peptide of the invention. In short, a virus inoculum is exposed to the peptide prior to infection of a permissive cell monolayer. After a standard interval the virus titer in the cellular extracts is determined using multiple dilutions of these extracts by infecting fresh cell monolayers and quantifying their effects on the cell monolayer.

For assessment of antiparasitic activity, a peptide of the invention and a parasite are incubated for a standard time interval. Thereafter, the metabolic activity of the parasites may be analyzed directly, for instance by an MTT assay, or the parasites are transferred to mammalian cells and after incubation parasite multiplication in these cells is assessed by microscopy.

The term "anti-inflammatory activity" of a peptide as used herein refers to inhibiting, reducing or preventing an inflammatory response, e.g. in a subject or in vitro, that has been infected by microbes, e.g. bacteria, viruses, fungi, and/or parasites. Anti-inflammatory activity is achieved by inhibiting, reducing or preventing the release, or by neutralization of pro-inflammatory microbial compounds, such as lipoteichoic acid (LTA), peptidoglycan (PG) and/or

lipopolysaccharides (LPS), and/or by microorganisms, such as S. aureus. Antiinflammatory activity can be measured by methods known in the art. Examples of such method are a LPS neutralization assay and a LTA neutralization assay. For instance, a peptide of the invention is mixed with a fixed concentration of LPS or LTA, such as 500 ng/ml LPS or 2 mg/ml LTA, and incubated for 30 min. Thereafter, these mixtures are added to diluted fresh human whole blood and after incubation the level of cytokines (e.g. IL-8 for LTA and S. aureus and IL- 12p40 for LPS) in the blood sample are measured by ELISA. Suitable methods are described in the Examples.

Preferred peptides of the invention have antimicrobial and/or antibiofilm activity in phosphate buffered saline (i.e. in the presence of

physiological concentrations of NaCl). "Phosphate buffered saline" or "PBS" as used herein refers to a buffer solution commonly used in the art. It is a water-based salt solution containing sodium chloride, sodium phosphate, and optionally potassium chloride and potassium phosphate and generally has a pH of 7.4. Methods for preparing PBS and concentrations of sodium chloride, sodium phosphate, and optionally potassium chloride and potassium phosphate, are well known in the art.

Even more preferred peptides of the invention are resistant to plasma, also referred to as blood plasma, preferably to human plasma. "Plasma" or "blood plasma" as used herein has the common meaning use in the art. It refers to the fluid portion of blood from which red and white blood cells and platelets are removed, which portion includes proteins, including coagulation factors, hormones and other organic compounds and inorganic compounds such as electrolytes.

"Resistance to plasma" as used herein is defined as having an in vitro

antimicrobial, anti-biofilm, antibacterial, antiviral, antifungal and/or antiparasitic activity against at least one microbial species in the presence of at least 50% plasma, preferably human plasma. With "in the presence of at least 50% plasma" is meant that the antimicrobial activity is measured when a peptide is incubated with microbes in for instance liquid such as PBS with addition of plasma, preferably human plasma, at a final concentration of 50%.

As detailed herein, the in vitro antimicrobial, antibacterial, antiviral, antifungal and/or antiparasitic activity is preferably expressed as the LC99.9.

"LC99.9" as used herein refers to the lowest peptide concentration which kills at least 99.9% of microbes within a given period, for instance 2 hours, 1 day or 48 hours. It is also possible to assess the lowest peptide concentration which kills at least another percentages of microbes, such as at least 50%, at least 90% or at least 99% of microbes. "Resistance to plasma" as used herein preferably refers to an in vitro LC99.9 that is at most 120 μΜ after 2 hours incubation of peptide with microbe in the presence of plasma at 37°C. Said LC99.9 is preferably determined in accordance with a method for determining the antibacterial or antifungal activity as described herein in the Examples. Said method described in the Examples for determining antibacterial or antifungal involves the incubation of a peptide with lxlO⁶ cfu/ml microbes, preferably bacteria or fungi in PBS with added pooled plasma at a final concentration of 50% for 2 hours at 37°C under shaking conditions. Plasma-resistant peptides of the invention preferably have an in vitro LC99.9 of at most 60 μΜ in the presence of (human) plasma or blood against at least one bacterial or fungal species, more preferably at most 30 μΜ, more preferably at most 15 μΜ. Said at least one microbial species is for instance a bacterial species such as S. aureus, B. subtilis, E. coli, S. epidermidis, E. faecium, K. pneumoniae, A. baumannii or P. aeruginosa, a fungal species such as C.

neoformans, C. albicans or A. niger, a parasitic species such as Plasmodium falciparum and Toxoplasma gondii, or a virus species such as hepatitis A virus, hepatitis C virus, Influenza A virus, etc. Preferably, a peptide of the invention has an in vitro LC99.9 as defined herein against at least one bacterial or fungal species in the presence of plasma or blood, preferably human plasma or blood, against at least one microbe selected from the group consisting of S. aureus and A. niger. Said S. aureus is preferably Staphylococcus aureus strain JAR060131, LUH 15095 or LUH 15096, MRSA LUH 14616 or MRSA LUH 15094.

A variant of amino acid sequence LRCMCIKWWSGKHPK has one or more of the following modifications:

substitution of up to four amino acids selected from the group consisting of L, C, M, I, W, T, S, G, H and P by an amino acid selected from the group consisting of K and R; substitution of up to 5 amino acid selected from the group consisting of L, C, M, I, W, T, S, G, H and P by another amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W and Y;

substitution of one or more of K by R and/or R by K;

Preferably said variant sequence has at most 4 of said modifications more preferably at most 3, more preferably at most 2, of said modifications.

Preferred peptides have a substitution of up to three amino acids selected from the group consisting of L, C, M, I, W, T, S, G, H and P by an amino acid selected from the group consisting of K and R, preferably by K, such as 3, 2 or 1 of said substitutions, as such substitution may increase antimicrobial activity as demonstrated in the Examples. Said variant sequence further preferably has up to 5 amino acids selected from the group consisting of L, C, M, I, W, T, S, G, H and P substituted by another amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W and Y, more preferably up to 4 amino acids selected from the group consisting of L, C, M, I, W, T, S, G, H and P, more preferably up to 3, such as 3, 2 or 1 of said substitutions. Preferably at least one of the amino acids at position 6 and 8 of sequence LRCMCIKWWSGKHPK are preserved in a peptide of the invention. A preferred peptide of the invention comprises at least 10 amino acids of amino acid sequence LRCMCIKWWSGKHPK or of a variant of said amino acid sequence with substitution of up to two amino acid selected from the group consisting of K and R by an amino acid selected from the group consisting of A, C, F, H, G, I, L, M, N, P, Q, S, T, V, W and Y, preferably A, and/or substitution of up to four amino acid selected from the group consisting of L, C, M, I, W, T, S, G, H and P by an amino acid selected from the group consisting of K and R. Insertion of amino acid is preferably of one amino acid selected from the group consisting of A, C, F, H, G, I, L, M, N, P, Q, S, T, V, W and Y and preferably is between amino acid W at position 9 and A at position 10 of the sequence LRCMCIKWWSGKHPK. Preferably an amino acid selected from the group consisting of W, Y and F is inserted between said positions 9 and 10 of the sequence. Addition of amino acids is preferably with one or two amino acids selected from the group consisting of A, C, F, H, G, I, L, M, N, P, Q, S, T, V, W and Y at the N-terminus of the sequence, more preferably with one or two additional amino acids selected from W, WW, F, FF, Y and YY at the N-terminus of the sequence, most preferably at the N-terminus of a variant comprising at least amino acids 1-10 of said sequence, preferably comprising all 15 amino acids of said sequence.

A peptide of the invention comprises at least 10 amino acids of the sequence LRCMCIKWWSGKHPK or at least 10 amino acids of a variant sequence thereof as defined herein. Preferably said peptide comprises at least 11 amino acids of said sequence or of said variant sequence, more preferably at least 12 amino acids of said sequence or of said variant sequence, more preferably at least 13 amino acids of said sequence or of said variant sequence, more preferably at least 14 amino acids of said sequence or of said variant sequence. A particularly preferred peptide of the invention comprises at least 15 amino acids of the sequence LRCMCIKWWSGKHPK or of a variant sequence thereof.

Provided is therefore synthetic, isolated or recombinant peptide of 10-25 amino acids comprising at least 10 amino acids of amino acid sequence

LRCMCIKWWSGKHPK, or at least 10 amino acids of a variant of said sequence, said peptide having antimicrobial, antibacterial, antiviral, antifungal, antiparasitic and/or anti-inflammatory activity; said variant sequence having one or more, preferably up to 3, of the following modifications:

substitution of one or both of C, or of K at position 12, or of L, or of I or one or both of W by an amino acid selected from the group consisting of A, C, F, H, G, I, L, M, N, P, Q, S, T, V, W and Y,

removal of one or both of C, or substitution of one or both of C by a negatively charged amino acid or a non-natural amino acid, preferably aminobutyric acid;

substitution of R by K and/or one or more of K by R;

substitution of H by K or R;

substitution of one amino acid at position 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, 14 or 15 by an amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W and Y; insertion of one amino acid selected from the group consisting of W, F and Y, between W at position 9 and S at position 10 in said sequence;

addition of one or two amino acids selected from W, WW, F, FF, Y and YY at the N-terminus of said sequence.

A preferred peptide comprises at least 10 amino acids, preferably 15 amino acids, of amino acid sequence LRCMCIKWWSGKHPK or of a variant of said sequence, said variant sequence having one or more, preferably up to 3, of the following modifications:

substitution of one of both of C by I or W;

substitution of K at position 12 by I or W;

substitution of L or I by W;

substitution of one or both of W by F or Y;

substitution of R by K and/or one or more of K by R;

substitution of H by K;

substitution of one amino acid at position 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, 14 or 15 by an amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W and Y, preferably by A;

insertion of one amino acid selected from the group consisting of W, F and Y between W at position 9 and S at position 10 in said sequence;

addition of one or two amino acids selected from W, WW, F, FF, Y and YY at the N-terminus of said sequence,

said peptide optionally comprising an N-terminal acetyl and/or a C-terminal amide.

Peptides having such sequence or variant thereof have been shown to retain antimicrobial activity in PBS or in the present of plasma in the Examples. Said peptide preferably consists of 10-15, more preferably 15 amino acids of said sequence, optionally with one or two additional amino acids selected from W, WW, F, FF, Y and YY at the N-terminus of said sequence.

A further preferred peptide of the invention comprises at least 10 amino acids, preferably 15 amino acids, of amino acid sequence LRCMCIKWWSGKHPK or of a variant of said sequence, said variant sequence having one of the following modifications: substitution of H by K or substitution of L at position 1, R at position 2, M at position 4, C at position 5, G at position 11, K at position 12 and P at position 14 by an amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W and Y, preferably by an amino acid selected from the group consisting of A, C, G, I, L, M, N,, P, Q, S, T and V, more preferably by A, said peptide optionally comprising an N-terminal acetyl and/or a C-terminal amide. Peptides having such sequence or variant thereof have been shown to retain antimicrobial activity in the present of plasma in the Examples. Said peptide preferably consists of 10-15, more preferably 15 amino acids of said sequence.

Preferred peptides in accordance with the invention comprise an amino acid sequence of a peptide depicted in Table 3 as having a LC99,9 of <60 μΜ in PBS or in 50% plasma because these peptides have antimicrobial activity in the biologically relevant media PBS and/or in plasma. Preferably, said peptide comprises an amino acid sequence of peptide TC19, TC37, TC43, TC57, TC63, TC69, TC70 or TC75 as depicted in Table 3, of peptide TC82, TC83, TC84, TC85, TC86, TC88, TC90, TC91, TC92, TC93, TC94, TC95 or TC96 as depicted in Table 7 or of peptide TC112, Tcll3, TC117, TC120, TC122, TC123, TC124, TC126, TC127, TC128, TC131, TC133 or TC166 as depicted in Table 8 because these peptides have particularly high antimicrobial activity against in PBS and/or in the presence of plasma. More preferably, said peptide comprises an amino acid sequence of peptide TC19, TC69 or TC75 as depicted in Table 3 because these peptides have a high antimicrobial activity in the presence of 50% plasma, indicating that these peptides are not degraded in the presence of plasma. Other preferred peptides have an amino acid sequence of peptides Al, E2, L3, R4, C5 as depicted in Table 1, or R4A, K9A, K17A, L3K, C5K, M6K, C7K, I8K, T10K, T11K, S12K, G13K, I14K, H15K or P16K as depicted in Table 2 because these peptides have broad spectrum

antimicrobial activity, at least against both bacteria and fungi.

A peptide according to the invention preferably comprises 10 amino acids of amino acid sequence LRCMCIKWWSGKHPK or of a variant sequence thereof selected from table 1 or 2 having an LC99.9 of at most 60 μΜ against at least one microbial species. Preferably, said sequence is selected from table 1, 2, 3, 5 or 8 and represents a peptide having an LC99.9 of at most 60 μΜ against at least one microbial species, more preferably at most 30 μΜ, more preferably at least 15 μΜ, more preferably at most 8 μΜ against at least one microbial species.

Particularly preferred peptides have a sequence as depicted in table 3, most preferably those sequences which represent a peptide having an LC99.9 of at most 60 μΜ against S. aureus in the presence of 50% plasma.

The invention further provides a synthetic, isolated or recombinant peptide according to the invention further optionally having an N-terminal and/or C-terminal modification, preferably comprising an N-terminal acetyl-, hexanoyl-, decanoyl-, myristoyl-, NH-(CH2-CH2-0)n-CO- or propionyl-residu and/or a C- terminal amide-, NH-(CH2-CH2-0)n-CO-amide-, or one or two amino-hexanoyl groups, and/or wherein said variant sequence optionally has one or more of the following modifications:

substitutions of an amino acid by a corresponding non-natural amino acid; substitutions of an amino acid by a corresponding D-amino acid.

Thus, in addition to substitutions of an amino acid by another amino acid as described above, a variant of amino acid sequence LRCMCIKWWSGKHPK as defined herein may contain one or more substitutions of an L- amino acid by its corresponding D-amino acid or by the D-amino acid corresponding to an L-amino acid that is present in said amino acid sequence after one or more of the amino acid substitutions indicated above. Amino acids indicated herein by an upper case single-letter symbol, such as A for alanine, are those L-amino acids commonly found in naturally occurring proteins. "Corresponding D-amino acid" as used herein is defined as the D-amino acid counter part of an L-amino acid. For examples, the corresponding D-amino acid of alanine (A) is D-alanine (a), the corresponding D-amino acid of arginine (R) is D-arginine (r), the corresponding D- amino acid of asparagine (N) is D-asparagine (n), etc. All L-amino acids of a variant sequence as defined herein can be substituted by their corresponding D-amino acids. A variant sequence as defined herein may contain up to 19 substitutions of an L-amino acid by its corresponding D-amino acid. Hence, the variant sequence may consist entirely of D-amino acids. For instance, the variant sequence may contain 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 5, 4, 3, 2 or 1 substitutions of an L-amino acid by its corresponding D-amino acid. Preferably, said variant sequence having one or more substitutions of an L-amino acid by its corresponding D-amino acid comprises the D-amino acids corresponding to the amino acids of peptides Al, E2, L3, R4 or C5 as depicted in Table 1 or of peptides R4A, K9A, K17A, L3K, C5K, M6K, C7K, I8K, T10K, T11K, S12K, G13K, I14K, H15K or P16K as depicted in Table 2, or of peptides TC19, TC37, TC43, TC57, TC63, TC69, TC70 or TC75 as depicted in Table 3 or of peptide TC82, TC83, TC84, TC85, TC86, TC88, TC90, TC91, TC92, TC93, TC94, TC95 or TC96 as depicted in Table 7. More preferably a peptide of the invention optionally comprises one or more substitutions of an L- amino acid of the amino acid sequence of peptide TC19 by its corresponding D- amino acid. In one embodiment, a variant sequence as defined herein contains one substitution of an amino acid by its corresponding D-amino acid. The position of the D-amino acid in the amino acid sequence is irrelevant. In another embodiment, the variant sequence contains substitution of all L-amino acids by their

corresponding D-amino acid.

A variant of amino acid sequence LRCMCIKWWSGKHPK as defined herein may comprise up to 5 substitutions of an amino acid by a non-natural amino acid, or of an amino acid that is present in said amino acid sequence after one or more of the amino acid substitutions indicated above by a non-natural amino acid. "Non-natural amino acids" as used herein refers to non- genetically encoded amino acids, irrespective of whether they appear in nature or not. Non-natural amino acids that can be present in a variant of an amino acid sequence as defined herein include: β-amino acids; p-acyl-L-phenylalanine; N-acetyl lysine; O-4-allyl-L- tyrosine; 2-aminoadipic acid; 3-aminoadipic acid; beta-alanine; 4-tert -butyl hydrogen 2-azidosuccinate; beta-aminopropionic acid; 2-aminobutyric acid; 4- aminobutyric acid; 2, 4,-diamino butyric acid; 6-aminocaproic acid; 2- aminoheptanoic acid; 2-aminoisobutyric acid ; 3-aminoisobutyric acid ; 2- aminopimelic acid; p-aminophenylalanine; 2, 3-diaminobutyric acid; 2, 3-diamino propionic acid; 2, 2'-diaminopimelic acid; p-amino-L-phenylalanine; p-azido-L- phenylalanine; D-allyl glycine; p-benzoyl-L-phenylalanine; 3-benzothienyl alanine p-bromophenylalanine; t-butylalanine; t-butylglycine; 4-chlorophenylalanine;

cyclohexylalanine; cysteic acid; D-citrulline; thio-L-citrulline; desmosine; epsilon- amino hexanoic acid; N-ethylglycine; N-ethylasparagine; 2-fluorophenylalanine; 3- fluorophenylalanine; 4-fluorophenylalanine; homoarginine; homocysteine;

homoserine; hydroxy lysine; alio- hydroxy lysine; 3-(3-methyl-4-nitrobenzyl)-L- histidine methyl ester; isodesmosine; allo-isoleucine; isopropyl-L-phenylalanine; 3- methyl-phenylalanine; N-methylglycine; N-methylisoleucine; 6-N-methyllysine; O- methyl-L-tyrosine; N-methylvaline; methionin sulfoxide; 2-napthylalanine; L-3-(2- naphthyl)alanine; isoserine; 3-phenylserine; norvaline; norleucine; 5,5,5-trifluoro- DL-leucine; ornithine; 3-chloro-tyrosine; N5 -carbamoylornithine; penicillamine; phenylglycine; piperidinic acid; pyridylalanine; 1, 2, 3, 4-tetrahydro-isoquinoline-3- carboxylix acid; beta-2-thienylalanine; γ-carboxy-DL-glutamic acid; 4-fluoro-DL- glutamic acid; D-thyroxine; allo-threonine; 5-hydroxy-tryptophan; 5-methoxy- tryptophan; 5-fluoro-tryptophan; 3-fluoro-valine.

Preferably, a natural amino acid of said sequence is substituted by a corresponding non-natural amino acid. As used herein, a "corresponding non- natural amino acid" refers to a non-natural amino acid that is a derivative of the reference natural amino acid. For instance, a natural amino acid is substituted by the corresponding β-amino acid, β-amino acids have their amino group bonded to the 6 carbon rather than the a carbon as in the natural amino acids. For instance, a-alanine is substituted by 6-alanine, etc. Other examples of substitution of a natural amino acid by a non-natural amino acid that is a derivative of said natural amino acid are the following. Alanine is for instance substituted by beta-alanine, t- butylalanine, 2-napthylalanine; L-3-(2-naphthyl)alanine, 2-aminoisobutyric acid. Arginine is for instance substituted by homoarginine, ornithine, N5- carbamoylornithine, 3- amino-propionic acid. Asparagine is for instance substituted by N-ethylasparagine. Aspartic acid is for instance substituted by 4-tert-butyl hydrogen 2-azidosuccinate. Cysteine is for instance substituted by cysteic acid, homocysteine. Glutamic acid is for instance substituted by γ-carboxy-DL- glutamic acid; 4-fluoro-DL-glutamic acid. Glutamine is for instance substituted by D- citrulline, thio-L-citrulline. Glycine is for instance substituted by N- methylglycine, t-butylglycine, N-methylglycine, D-allylglycine. Histidine is for instance

substituted by 3-(3-methyl-4-nitrobenzyl)-L-histidine methyl ester. Isoleucine is for instance substituted by isodesmosine, N-methylisoleucine, allo-isoleucine. Leucine is for instance substituted by norleucine, desmosine, 5,5,5-trifluoro-leucine. Lysine is for instance substituted by 6-N-methyllysine, 2-aminoheptanoic acid, N-acetyl lysine, hydroxylysine, allo-hydroxylysine. Methionine is for instance substituted by methionin sulfoxide. Phenylalanine is for instance substituted by p-amino-L- phenylalanine, 3-benzothienyl alanine p-bromophenylalanine, p-acyl-L- phenylalanine, 2-fluorophenylalanine, 3- fluorophenylalanine, 4- fluorophenylalanine. Proline is for instance substituted by 3-hydroxyproline, 4- hydroxyproline, l-acetyl-4-hydroxy-L-proline. Serine is for instance substituted by homoserine, isoserine, 3-phenylserine. Threonine is for instance substituted by D- thyroxine, allo-threonine. Tryptophan is for instance substituted by 5-hydroxy- tryptophan, 5-methoxy-tryptophan, 5-fluoro-tryptophan. Tyrosine is for instance substituted by O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 3-chloro-tyrosine. Valine is for instance substituted by norvaline, N-methylvaline, 3-fluoro-valine.

Also provided are peptoids having the amino acid sequence of a peptide of the invention or of a variant thereof as defined herein. The term "peptoid" as used herein refers to a peptidomimetic whose side chains are appended to the nitrogen atom of the peptide backbone, instead of to the a-carbons.

A peptide according to the invention may be N-terminally and/or C- terminally modified. Such N- or C-terminally modified peptide of the invention preferably comprises an N- and/or C-terminal elongating group. N- and C-terminal elongating groups that can be used in a peptide of the invention are well known in the art. Preferred examples of an N-terminal modification are an acetyl-, a hexanoyl-, a decanoyl-, a myristoyl-, a NH-(CH2-CH2-0)n-CO- and a propionyl- residu. Preferred examples of a C-terminal modification are an amide-, a NH-(CH2- CH2-0)n-CO-amide-, and one or two amino-hexanoyl groups. However, other N- or C-terminal elongating groups will also yield active compounds which is known to a person skilled in the art. Provided is thus a peptide according to the invention comprising an N-terminal acetyl-, hexanoyl-, decanoyl-, myristoyl-, NH-(CH2-CH2- 0)ii-CO- or propionyl-residu and/or a C-terminal amide-, NH-(CH2-CH2-0)n-CO- amide-, and one or two amino-hexanoyl groups.

A peptide according to the invention of 10-25 amino acids may consist of at least 10 amino acids from amino acid sequence LRCMCIKWWSGKHPK or of at least 10 amino acids of a variant of one of these sequences as defined herein, optionally with one or two additional N-terminal amino acid selected from the group consisting of W, F and Y. As used herein a "peptide" refers to peptides and peptidomimetics that comprise multiple amino acids. However, the amino acid sequence or variant thereof can be part of a larger peptide, i.e. of a peptide that has been N terminally and/or C-terminally extended by a one or more additional amino acids provided that the peptide does not comprise more than 25 amino acids. The advantages of small peptides of at most 25 residues over larger polypeptides and proteins are many and include the lowers costs and relative ease of large scale chemical synthesis of peptides up to approximately 50 residues, better tissue penetration and lower immunogenicity. The amino acid sequence or variant thereof of a peptide of the invention may be N-terminally and/or C-terminally modified, preferably by comprising an N- and/or C-terminal elongating group as described herein before. Alternatively, said amino acid sequence or a variant thereof is N- and/or C-terminally extended. As detailed in the Examples (Table 3), N terminal extension with one or more amino acids such as W, F or Y does not impair antimicrobial activity. A peptide according to the invention therefore comprises at least 10 amino acids, and may comprise up to 25 amino acids. Preferably, a peptide according to the invention is 10-25 amino acids in length, more preferably 10-20, amino acids, most preferably 10-15 amino acids. For instance, a peptide according to the invention comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. In one embodiment, a peptide of the invention consists of 10- 15 amino acids from amino acid sequence LRCMCIKWWSGKHPK or of 10-15 amino acids of a variant of one of these sequences as defined herein, optionally having an N-terminal and/or C-terminal modification as defined herein. In one embodiment, a peptide of the invention consists of 15 amino acids from amino acid sequence LRCMCIKWWSGKHPK or of 15 amino acids of a variant of one of these sequences as defined herein, optionally having an N-terminal and/or C-terminal modification as defined herein.

Provided is therefore a peptide consisting of 10-15 amino acids, comprising at least 10 amino acids of amino acid sequence LRCMCIKWWSGKHPK or of a variant of said sequence, said variant sequence having one or more, preferably up to 3, of the following modifications:

substitution of one of both of C by I or W;

substitution of K at position 12 by I or W;

substitution of L or I by W;

substitution of one or both of W by F or Y; substitution of R by K and/or one or more of K by R;

substitution of H by K;

substitution of one amino acid at position 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, 14 or 15 by an amino acid selected from the group consisting of A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W and Y;

As used herein, "peptidomimetic" refers to a compound containing non- peptidic structural elements which compound mimics the antimicrobial, antibacterial, antiviral, antifungal, antiparasitic and/or anti-inflammatory properties of a peptide of the invention. Hence, a peptide of the invention may comprise non-peptidic structural elements. Such non-peptidic structural elements may be present in the amino acid sequence of a peptide of the invention as a result of substitution of modification of one or more amino acids of said sequence.

Alternatively, a peptide of the invention may comprise non-peptidic structural elements outside the amino acid sequence LRCMCIKWWSGKHPK, or a variant thereof as defined herein, i.e. in the optional N- and/or C-terminal elongating groups. A non-peptidic structural element in a peptidomimetic is typically a modification of one or more existing amino acids. Preferred peptidomimetics are obtained by structural modification of peptides of the invention, for instance using unnatural amino acids such as defined herein above, conformational restraints, cyclization of the peptide, isosteric replacement or other modifications. The amino acid sequence of a peptide according to the invention thus optionally comprises one or more modifications. Such peptide may be modified by natural processes, such as posttranslational processing, or by chemical modification techniques. Modifications may be inserted at any location in said peptide, including in the peptide backbone, amino acid side-chains and at the N- or C-terminus. A single peptide may contain multiple types of modifications or several modification of a single type.

Modifications include acetylation, amidation, acylation, phosphorylation, methylation, demethylation, ADP-ribosylation, disulfide bond formation, ubiquitination, gamma-carboxylation, glycosylation, hydroxylation, iodination, oxidation, pegylation and sulfation. In addition a peptide according to the invention may be provided with a label, such as biotin, fluorescein or flavin, a lipid or lipid derivative, a sugar group. A peptide according to the invention can further be provided with a targeting moiety.

In a preferred embodiment, a polypeptide according to the invention comprises a cell penetrating peptide. Such cell penetrating peptide is a peptide sequences that, when linked to a antimicrobial peptide of the invention, facilitate efficient translocation of the polypeptide across cell membranes. Any cell penetrating peptide known in the art can be used in a polypeptide of the invention. Examples of cell penetrating peptides include, but are not limited to, polyarginine, TAT, HIV-Tat, R9-TAT, Pep- 1, Pep-7, penetratin, transportan, Antp, Rev, FHV coat protein, buforin II, MAP, K-FGF, Ku70, SynBl, HN-1, TP10, pVEC, BGSC, and BGTC. Preferred cell-penetrating peptides are peptides comprising or having the amino acid sequence ARKKAAKAARKKAAKAGG, PLIYLRLLRGQFAGG, PRRPRRPRRGG, RQIKIWFQNRRMKWKKGG or RWRRWWRRWGG.

A peptide of the invention is preferably a peptide that does not occur as such in nature. I.e. a peptide of the invention is preferably a non-naturally occurring peptide. "Non-naturally occurring" as used herein means that the peptide is not found in nature in that form, preferably that the amino acid sequence of the peptide is not found in nature.

Salts of peptides according to the invention are also provided. Such salts include, but are not limited to, acid addition salts and base addition salts. As used herein, "pharmaceutically acceptable salt" of a peptide refers to a salt that retains the desired antimicrobial, antibacterial, antifungal, antiviral, antiparasitic and/or anti-inflammatory activity of the peptide, and is suitable for administration to humans or animals. Methods for the preparation of salts of peptides are known in the art and generally involve mixing of the peptide with a pharmaceutically acceptable acid or based, for instance by reacting the free acid or free base forms of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze -drying, or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin. Examples of pharmaceutically acceptable acids and bases include organic and inorganic acids such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoro acetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid, and thiocyanic acid, which form ammonium salts with free amino groups of peptides, and bases which form carboxylate salts with free carboxylic groups of peptides, such as ethylamine, methylamine, dimethylamine,

triethylamine, isopropylamine, diisopropylamine, and other mono-, di-and trialkylamines, and arylamines.

Peptides according to the invention can be prepared by various methods. For instance, a peptide can be synthesized by commonly used solid-phase synthesis methods, e.g. methods that involve t-BOC or FMOC protection of alpha- amino groups which are well known in the art. Here, amino acids are sequentially added to a growing chain of amino acids. Such methods are for instance described in Merrifield (1963), J. Am. Chem. Soc. 85: 2149-2156 ; and Atherton et al, "Solid Phase Peptide Synthesis," IRL Press, London, (1989). Solid-phase synthesis methods are particularly suitable for synthesis of peptides or relatively short length, such as peptides of the invention with a length of up to 25 amino acids in large-scale production.

Alternatively, a peptide of the invention can be prepared using recombinant techniques well known in the art in which a nucleotide sequence encoding the peptide is expressed in host cells. The invention thus provides a method for the preparation of a peptide according to the invention comprising: providing a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide according to the invention;

transforming a host cell with said nucleic acid molecule;

culturing said host cell under conditions that allow expression of said peptide; harvesting said peptide from said cells.

The invention further provides a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide according to the invention, which is herein also referred to as a nucleic acid molecule according to the invention. As used herein, a nucleic acid molecule or nucleic acid sequence of the invention comprises a chain of nucleotides, preferably DNA and/or RNA. Further provided is a vector comprising a nucleic acid sequence molecule according to the invention. The term "vector" as used herein refers to a nucleic acid molecule, such as a plasmid, bacteriophage or animal virus, capable of introducing a heterologous nucleic acid sequence into a host cell. A vector according to the invention allows the expression or production of a peptide of the invention encoded by the heterologous nucleic acid sequence in a host cell. A vector used in accordance with the invention is for instance derived from an animal virus, examples of which include, but not limited to, vaccinia virus (including attenuated derivatives such as the Modified Vaccinia virus Ankara, MVA), Newcastle Disease virus (NDV), adenovirus or retrovirus. A vector according to the invention preferably comprises an expression cassette comprising a promoter that is suitable for initiation of transcription of a peptide according to the invention in the selected host cells. Examples of suitable promoters for expression of peptides according to the invention in eukaryotic host cells include, but are not limited to, beta-actin promoter, immunoglobin promoter, 5S RNA promoter, or virus derived promoters such as cytomegalovirus (CMV), Rous sarcoma virus (RSV) and Simian virus 40 (SV40) promoters for mammalian hosts. Further provided by the invention is a recombinant host cell comprising a nucleic acid molecule and/or a vector according to the invention. A host cell is a cell which has been transformed, or is capable of transformation, by a nucleic acid molecule such as a vector according to the invention. "Transformation" refers to the introduction of a foreign nucleic acid into a recipient cell. Transformation of a host cell can result in transient expression of a recombinant protein by said cell, meaning that the recombinant protein is only expressed for a defined period of time. Alternatively, transformation of a recipient cell can result in stable expression, meaning that the nucleic acid is introduced into the genome of the cell and thus passed on to next generations of cells. Additionally, inducible expression of a recombinant protein can be achieved. An inducible expression system requires the presence or absence of a molecule that allows for expression of a nucleic acid sequence encoding a peptide of the invention. Examples of inducible expression systems include, but are not limited to, Tet-On and Tet-Off expression systems, hormone inducible gene expression system such as for instance an ecdysone inducible gene expression system, an arabinose-inducible gene expression system, and a Drosophila inducible expression system using a pMT/BiP vector (Invitrogen) which comprises an inducible metallothioneine promoter. A host cell used in a method for the preparation of a peptide according to the invention is for instance a Gram-positive prokaryote, a Gram-negative prokaryote or an eukaryote. Preferably said host cell is an eukaryotic cell, such as a plant cell, a yeast cell, a mammalian cell or an insect cell, most preferably an insect cell or a mammalian cell. Examples of suitable host cells include plant cells such as corn cells, rice cells, duckweed cells, tobacco cells (such as BY-2 or NT-1 cells), and potato cells. Examples of yeast cells are Saccharomyces and Pichia. Examples of insect cells are Spodoptera frugiperda cells, such as Tn5, SF-9 and SF-21 cells, and Drosophila cells, such as Drosophila Schneider 2 (S2) cells. Examples of mammalian cells that are suitable for expressing a peptide according to the invention include, but are not limited to, African Green Monkey kidney (Vero) cells, baby hamster kidney (such as BHK-21) cells, Human retina cells (for example PerC6 cells), human embryonic kidney cells (such as HEK293 cells), Madin Darby Canine kidney (MDCK) cells, Chicken embryo fibroblasts (CEF), Chicken embryo kidney cells (CEK cells), blastoderm- derived embryonic stem cells (e.g. EB14), mouse embryonic fibroblasts (such as 3T3 cells), Chinese hamster ovary (CHO) cells , and derivatives of these cell types. A method according to the invention preferably further comprises a step of

harvesting, purifying and/or isolating peptides according to the invention. Obtained peptides according to the invention are preferably used in human therapy, optionally after additional purifying, isolation or processing steps, for instance purification using gel electrophoresis or chromatography methods.

A peptide according to the invention exhibits a number of activities that can be advantageously used in both therapeutic and nontherapeutic applications. In particular, peptides according to the invention are useful in counteracting various microbial infections, such as bacterial infections, fungal infections, viral infections, and in counteracting parasitic infections. In addition, preferred peptides have anti-inflammatory activity. Provided are thus pharmaceutical compositions comprising a peptide according to the invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent and/or excipient. Also provided are pharmaceutical compositions comprising a nucleic acid molecule or vector according to the invention and at least one pharmaceutically acceptable carrier, diluent and/or excipient. Pharmaceutical compositions according to the invention include both compositions for human use and compositions for veterinary use.

The invention further provides a peptide according to the invention for use as a medicament. Further provided is a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide according to the invention for use as a medicament. Said medicament can be a therapeutic or a prophylactic agent.

In one embodiment, the invention provides a method for the treatment of a subject suffering from or at risk of suffering from a bacterial, fungal, viral and/or parasitic infection comprising administering to said subject a

therapeutically effective amount of a peptide according invention, a pharmaceutical composition according to the invention or a nucleic acid molecule according to the invention. Also provided is a method for the preparation of a medicament for the treatment of a subject infected with a microbe or for prophylaxis of a microbial infection. In a preferred embodiment, said microbe is a bacterium, a fungus, a virus or a parasite. Further provided is a peptide and/or nucleic acid molecule for use according to the invention in the prevention or treatment of a microbial, bacterial, fungal, viral and/or parasitic infection or a condition resulting from a microbial, bacterial, fungal, viral and/or parasitic infection.

As used herein, an "subject" is a human or an animal. Subjects include, but are not limited to, mammals such as humans, pigs, ferrets, seals, rabbits, cats, dogs, cows and horses, and birds such as chickens, ducks, geese and turkeys. In a preferred embodiment of the invention a subject is a mammal. In a particularly preferred embodiment the subject is a human.

The invention also provides a method for inhibiting the growth of a microbe, e.g. a bacterium, a virus, a fungus, or a parasite comprising contacting said microbe or parasite with a peptide or pharmaceutical composition according to the invention. Said contacting can be performed in vivo and in vitro.

The peptides and pharmaceutical compositions according to the invention are effective in treating a variety of microbial infections, such as various viral, bacterial and fungal infections. For example, the peptides and

pharmaceutical compositions are effective in treating Gram-negative and Gram- positive bacteria. Examples of pathogenic bacteria that may cause infections in humans or animals that are treatable with peptides and compositions of the invention include, but are not limited to, Listeria, Escherichia, Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptocci, pneumonococci, meningococci, Klebsiella, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Vibrio cholerae, Clostridium tetani, Bacillus species, Yersinia,

Enterococcus bacteria, Acinetobacter species and Leptospira bacteria.

Examples of pathogenic viruses that may cause infections in humans or animals that are treatable with peptides and compositions of the invention include, but are not limited to, A, B or C hepatitis, herpes virus (for instance VZV, HSV-I, HAV-6, HSV-II, CMV, EpsteinBarr-virus), adenovirus, influenza virus,

flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus (RSV), rotavirus, Morbillivirus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, poliovirus, rabies virus and human immunodeficiency virus (HIV virus; e. g., type I and II).

Examples of pathogenic fungi that may cause infections in humans or animals that are treatable with peptides and compositions of the invention include, but are not limited to, Candida (e.g., albicans, krusei, glabrata, tropicalis),

Aspergillus (e.g., fumigatus, niger), Cryptococcus neo for mans, Histoplasma capsulatum, Genus Mucorales, Blastomyces dermatitidis, Paracoccidioides brasiliensis , and Coccidioides immitis.

Examples of pathogenic parasites that may cause infections in humans or animals that are treatable with peptides and compositions of the invention include, but are not limited to, Entamoeba histolytica, Plasmodium (e.g.

falciparum, vivax), Entamoeba, Giardia, Balantidium coli, Acanthamoeba,

Cryptosporidium sp., Pneumocystis carinii, Babesia microti, Trypanosoma (e.g. brucei, cruzi), Leishmania (e.g. donovani), and Toxoplasma gondii.

In preferred embodiment, peptides and pharmaceutical compositions of the invention are effective in treating infections caused by Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii and/or Cryptococcus neoformans.

The compositions containing the peptides can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, peptides or compositions are administered to a subject, preferably a human, already suffering from a disease in an amount sufficient to counteract the symptoms of the infection or the condition resulting from the infection and its complications. In prophylactic applications, peptides or compositions are administered to a subject, for instance a human or animal at risk of suffering from a microbial or parasitic infection in an amount sufficient to prevent infection or at least inhibit the development of an infection. The peptide is typically present in a pharmaceutical composition according to the invention in a therapeutic amount, which is an amount sufficient to remedy a condition or disease, particularly symptoms associated with a microbial or parasitic infection. Typical doses of administration of a peptide according to the invention or combinations of at least two thereof are between 0.01 and 10 mg peptide per kg body weight, depending on the size of the peptide.

For instance, peptides of the invention are particularly suitable used as a preservative for materials that are susceptible to microbial, e.g. bacterial, viral, fungal, parasitic, infection. Such material can be impregnated or coated with or covered by a peptide of the invention. As detailed herein before, peptides of the invention are retain antimicrobial activity in blood, plasma and serum, and in the presence of components, such as plasma components. Peptides and pharmaceutical composition of the invention are therefore particularly suitable for systemic application and for treatment and/or prevention of infection associated with biomaterials, implants and medical devices. The term "biomaterial" as used herein is refers to any material, natural or synthetic, that is introduced on or in the human or animal body, and included medical devices and implants. The term "medical devices" as used herein refers to any type of device that can be used in the human or animal body and includes, but is not limited to, medical instruments, medical implements, prostheses, such as artificial joints including hips and knees, and dental prostheses, fracture fixation devices, breast implants, implantable devices such as pacemakers, heart valves, stents, catheters, ear tubes, splints, screws for medical devices, and wound or tissue dressings. Implants and other medical devices are often associated with microbial infection, in particular with biofilm infections. Preferred peptides of the invention are capable of preventing biofilm formation and disperse existing biofilms, kill bacteria, fungi or other microbes at and around the site of biofilm formation and more preferably also neutralize inflammatory components of microbial cells. Biomaterials, implants and medical devices are generally rapidly covered by plasma components from the hosts fluids after implantation. Because the peptides of the invention retain

antimicrobial activity in the presence of plasma components, microbial infection of implants and/or medical devices is effectively treated and/or prevented by a peptide according to the invention. Provided is therefore the use of a peptide of the invention to prevent infection of an implant and/or medical device. Also provided is a peptide of the invention for use in prevention and/or treatment of microbial infection, preferably bacterial infection, of an implant and/or medical devices.

A peptide of the invention is advantageously incorporated in a controlled release and/or targeted delivery carrier. As used herein, the term

"controlled release" refers to the release of the peptide of the invention in time dependent manner. In one embodiment, controlled release refers to slow release. As used herein, the term "targeted delivery" refers to the release of the peptide of the invention in a site-directed manner. Use of a controlled release vehicle has the advantage that frequent administration such as by injection of the peptide of the invention can be avoided. Use of a targeted delivery vehicle has the advantage that the peptide of the invention is effectively delivered to and/or retained at a site of interest in a subject's body, such as a site of inflammation or a site of infection. Preferably, a peptide of the invention is targeted to a site infected by

microorganisms including bacteria, fungi, viruses and parasites. Controlled release and/or targeted delivery carriers are well known in the art. Non limiting examples of controlled release and/or targeted delivery vehicles are nanoparticles,

microp articles, nanocapsules, microcapsules, liposomes, microspheres, hydrogels, polymers, lipid complexes, serum albumin, antibodies, cyclodextrins and dextrans. Controlled release is for instance provided by incorporating a peptide of the invention in or on the surface of such carrier. The carriers are of materials that form particles that capture a peptide of the invention and slowly degrade or dissolve in a suitable environment, such as aqueous, acidic or basic environment or body fluids, and thereby release the peptide. Targeted delivery is for instance achieved by providing a carrier with targeting groups on the surface thereof.

Examples of such carrier comprising targeting groups are antibody-functionalized carriers, carriers having a site-specific ligand and carriers having a positive or negative surface charge. Preferred particles for controlled release and/or targeted delivery are nanoparticles, i.e., particles in the range of about 1 to 500 nm in diameter, preferably up to about 200 nm in diameter, and liposomes, optionally provided with targeting groups. The invention therefore provides a controlled release carrier comprising a peptide of the invention and pharmaceutical compositions comprising such controlled release carrier. Also provided is a targeted delivery carrier comprising a peptide of the invention, and a pharmaceutical composition comprising such targeted delivery carrier. Said carrier is preferably selected from the group consisting of nanoparticles, microp articles, nanocapsules, microcapsules, liposomes, microspheres, hydrogels, polymers, lipid complexes, serum albumin, antibodies, cyclodextrins and dextran.

Preferred targeted delivery and/or controlled release carriers are of biodegradable material. "Biodegradable" as used herein refers to molecules that degrade under physiological conditions. This includes molecules that are

hydrolytically degradable and molecules that require enzymatic degradation.

Suitable biodegradable materials include, but are not limited to, biodegradable polymers and natural biodegradable material such as PLA (poly lactic acid), PGA (poly glycolic acid), polycaprolactone (PCA), polyvinylalcohol (PVA), polyethylene oxide (PEO), polydioxanone (PDS), polycaprolactone (PCL), polypropylene fumarate, polymers derived from lactones, such as lactide, glycolide and

caprolactone, carbonates such as trimethylene carbonate and tetramethylene carbonate, dioxanones, ethylene glycol, polyester amide (PEA) ethylene oxide, esteramides, γ-hydroxyvalerate, β-hydroxypropionate, a-hydroxy acid,

hydroxybuterates,hydroxy alkanoates, polyimide carbonates, polyurethanes, polyanhydrides, and combinations thereof, polysaccharides such as hyaluronic acid, chitosan and cellulose, and proteins such as gelatin and collagen. Coupling to self- assembling monomers, which can in situ form nano- or microagregates such as, but not limited to fibrils.

Further provided is a coating, preferably for biomaterials, implants and/or medical devices, comprising a peptides of the invention. In one embodiment, such coating provides for controlled release of the peptide of the invention. Such controlled release coating for medical devices preferably comprises a biodegradable material so that release of the peptide of the invention is achieved by degradation of the coating material. Also provided is therefore a controlled release coating comprising a peptide of the invention. Further provided is a biomaterial, medical device and/or implant provided with a coating according to the invention, preferably comprising a peptide of the invention and a biodegradable material. A biodegradable coating in accordance with the invention comprises a biodegradable material as defined above. In particular, such biodegradable coating comprises a material selected from the group consisting of PLA (poly lactic acid), PGA (poly glycolic acid), polycaprolactone (PCA), polyvinylalcohol (PVA), polyethylene oxide (PEO), polydioxanone (PDS), polycaprolactone (PCL), polypropylene fumarate, polymers derived from lactones, such as lactide, glycolide and caprolactone, carbonates such as trimethylene carbonate and tetramethylene carbonate, dioxanones, ethylene glycol, polyester amide (PEA) ethylene oxide, esteramides, γ- hydroxyvalerate, β-hydroxypropionate, a-hydroxy acid, hydroxybuterates,hydroxy alkanoates, polyimide carbonates, polyurethanes, polyanhydrides, and

combinations thereof, polysaccharides such as hyaluronic acid, chitosan and cellulose, proteins such as gelatin and collagen, and biodegradable polyesters and lipids such as polylactic-co- glycolic acid (PLGA), dipalmitoyl phosphatidyl choline (DPPC), distearoyl phosphatidyl choline (DSPC) and cholesterol, or the

combination of PLGA, DPPC, DSPC and cholesterol. Further, provided is a method of preventing and/or treating of microbial infection, preferably bacterial or fungicidal infection, of a biomaterial, implant and/or medical device comprising providing said biomaterial, implant and/or medical device with a coating comprising a peptide of the invention and introducing said biomaterial, implant or medical device in a subject. Peptides of the invention are also suitably used for topical application, e.g. in the treatment or prevention of skin infections, wound infections and urinary tract infections. Bacterial biofilms may delay cutaneous wound healing and reduce topical antibacterial efficiency of conventional antibiotics in healing or treating infected skin wounds, skin infections or urinary tract infections. The invention therefore provides a peptide, pharmaceutical composition and/or nucleic acid molecule according to the invention for use in the treatment or prevention of skin infection, wound infection and/or urinary tract infections. Also provided is a peptide, pharmaceutical composition and/or nucleic acid molecule according to the invention for use in would healing. Further provided is the use of a peptide, pharmaceutical composition and/or nucleic acid molecule according to the invention in the manufacture of a pharmaceutical composition for the treatment or prevention of skin infection, wound infection, urinary tract infection and/or for wound healing. The invention further provides a method for the treatment of a subject suffering from skin infection, wound infection and/or urinary tract infection comprising administering to said subject a therapeutically effective amount of a peptide according invention, a pharmaceutical composition according to the invention or a nucleic acid molecule according to the invention.

The peptides and pharmaceutical compositions are also useful as antiinflammatory agents, e.g. by neutralizing pro-inflammatory microbial endotoxins such as lipoteichoic acid, peptidoglycan and lipopolysaccharides either as released compounds or as part of the bacteria, thereby inhibiting, reducing or preventing influx of neutrophils, macrophages/monocytes and lymphocytes and the release of pro-inflammatory microbial compounds by the infected subject. Also provided is therefore a method for inhibiting the release of pro-inflammatory compounds comprising contacting a cell capable of releasing pro-inflammatory compounds with a peptide according to the invention. Said contacting can be performed in vivo and in vitro. Further provided is a peptide according to the invention for use as an antiinflammatory agent.

Peptides according to the invention are potent antimicrobial agents as such, but they can also be combined with known antimicrobial agents, such as conventional anti-infectives, such as antibiotics, antivirals and antifungals or other antimicrobial peptides, and antibodies and chemicals e.g. sensitizers, nano- particles. Such combination may result in an increased antimicrobial activity or broaden the spectrum of activity. Peptides of the invention may for instance be combined with penicillins, cephalosporins, macrolides, fluoroquinolones,

sulfonamides, tetracylcines and/or aminoglycosides for treating bacterial infections. For treatment of viral infections peptides may be combined with antiviral nucleoside analogs such as aciclovir, ganciclovir, zidovudine (AZT) or didanosine or neuramidase inhibitors such as oseltamivir, peramivir or zanamivir. For treatment of fungal infections the peptides and compositions of the invention may be combined with polyene antifungals, imidazoles, triazoles, allylamines,

echinocandins, ciclopirox, flucytosine and/or griseofulvin. The invention therefore provides a pharmaceutical composition comprising a peptide according to the invention and an additional antimicrobial agent, such as a antibiotic or an antimicrobial peptide, preferably selected from the group consisting of penicillins, cephalosporins, carbapenems and mupirocin.

Pharmaceutical compositions according to the invention comprise at least one pharmaceutically acceptable carrier, diluent or excipient. Examples of suitable carriers for instance comprise keyhole limpet haemocyanin (KLH), serum albumin (e.g. BSA or RSA) and ovalbumin. In a preferred embodiment said suitable carrier is a solution, for example saline. Examples of excipients which can be incorporated in tablets, capsules and the like are the following: a binder such as gum tragacanth, acacia, corn starch or gelatine; an excipient such as

microcrystalline cellulose; a disintegrating agent such as corn starch,

pre gelatinized starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen or cherry. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as fatty oil. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor. A

pharmaceutical composition according to the invention is preferably suitable for human use.

The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include administering a pharmaceutical composition comprising a peptide according to the invention and containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical,

intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal,

transdermal, intrathecal, and intracranial methods. For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.

Sterile compositions for injection can be formulated according to conventional pharmaceutical practice by dissolving or suspending the peptide of the invention in a vehicle for injection, such as water or a naturally occurring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate or the like. Buffers, preservatives, antioxidants and the like may also be incorporated.

Compositions for topical administration can also be formulated according to conventional pharmaceutical practice. "Topical administration" as used herein refers to application to a body surface such as the skin or mucous membranes to locally treat conditions resulting from microbial or parasitic infections. Examples of formulations suitable for topical administration include, but are not limited to a cream, gel, ointment, lotion, foam, suspension, spray, aerosol, powder aerosol. Topical medicaments can be epicutaneous, meaning that they are applied directly to the skin. Topical medicaments can also be inhalational, for instance for application to the mucosal epithelium of the respiratory tract, or applied to the surface of tissues other than the skin, such as eye drops applied to the conjunctiva, or ear drops placed in the ear. Said pharmaceutical composition formulated for topical administration preferably comprises at least one

pharmaceutical excipient suitable for topical application, such as an emulgent, a diluent, a humectant, a preservatives, a pH adjuster and/or water. Another useful application of peptides according to the invention is in preservation of food products. Also provided is therefore the use of a peptide according to the invention as a food preservative. Generally, pathogenic or spoilage microorganism are destroyed by thermally processing foods by subjecting them to temperatures varying from 60 to 100 °C. Such treatment may have undesirable effects on the food product, such as undesirable organoleptic effects. Use of a peptide according to the invention as a preservative in food products may result in extended storage life and/or enhanced safety of the food product. Pathogenic microorganisms in foods may cause infections or intoxication of subjects, and include bacteria such as Campylobacter jejuni, Salmonella typhi, Salmonella paratyphi and non-typhi Salmonella species, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Shigella, Bacillus cereus and Clostridium botulinum, viruses such as Rotaviruses and Norwalk virus, parasites such as Taenia solium, Taenia saginata and Trichinella spiralis and moulds. Food spoilage refers to the change of look, consistency, flavor and/or odor of food products, and may be caused by bacteria such as Lactobacillus, Leuconostoc, Pseudomonas, Micrococcus, Flavobacterium, Serratia, Enterobacter and Streptococcus, fungi such as

Aspergillus, Fusarium and Cladosporium and yeasts.

The invention will be explained in more detail in the following, non- limiting examples.

Brief description of the drawings

Figure 1: Sequence comparison of thrombocidins (TC- 1 and TC-2), neutrophil activating peptide-2 (NAP-2) and connective tissue activating peptide-III (CTAP- III). Synthetic peptides are presented schematically with only N- and C-terminal residues given. Code names of peptides are given in brackets. The positively charged lysine (K) and arginine (R) residues are shaded.

Figure 2: Microbicidal activity of thrombocidin-derived peptides. Inocula of 1-2 x 10⁵ cfu/ml of the indicated organisms were exposed to 60 μΜ of each peptide in 10 mM phosphate buffer + 0.06% TSB pH7.0. After 2 h of incubation microbial survival was determined by quantitative plating.

Figure 3: Inhibition of growth of Aspergillus niger growth by different

concentrations of peptide TC19 in PBS. Antifungal caspofungin (Cancidas)is used as a positive control. Values are expressed as

optical density at 600 nm relative to the optical density at 0 h.

Figure 4: Inhibition of Aspergillus niger growth by different concentrations of peptide TC19 in the presence of 25% plasma. Antifungal caspofungin (Cancidas) is used as a positive control. Representative light micrographs of triplicates.

Figure 5: Inhibition of biofilm formation by S. aureus JAR060131 at different concentrations (in μΜ) of peptide TC19. Results are expressed as mean percentage biofilm mass relative to the untreated sample (0) ± standard deviations of three independent experiments. (A) antibiofilm formation in BM2 medium in percentage. (B) and (C) Antibiofilm formation in BM2 medium and in plasma expressed in relative OD at 595 nm (B) and in percentage (C).

Figure 6. S. aureus killing kinetics of TCI 9

Figure 7: Immunomodulatory activity of TC19. (A) LPS neutralizing activity measured as inhibition of LPS-induced IL- 12p40 production by blood cells. (B) S. aureus neutralizing activity measured as inhibition of S. aureus-induced IL-8 production by blood cells.

Figure 8: Hemolytic activity of TC19 in PBS (A) and in 50% plasma (B).

Examples Materials and methods

Peptide synthesis

Peptides were synthesized on an Abimed 422 multiple peptide synthesizer

(Abimed, Langenfeld, Germany) at 10 μηιοΐ scale (23, 24). TentagelS AC resins (Rapp, Tiibingen, Germany) (Rapp, W, et al. 1990. Continuous flow peptide synthesis on PSPOE-graft copolymers, p. 205. In R. Epton (ed.), Innovation and perspectives in solid phase peptide synthesis. SPCC Ltd., Birmingham; Sheppard, RC and BJ Williams. 1982. Acid-labile resin linkage agents for use in solid phase peptide synthesis. Int.J.Peptide protein Res. 20:451) were used in combination with Fmoc-protected amino acids, carrying TFA-labile side chain protecting groups where needed (19). Acylations were carried out with a six-fold excess amino acid using PyBOP/NMM activation in NMP (12, 24). Deprotection was performed with piperidine/N,N-dimethylacetamide 1/4 (v/v). Cleavage of the peptides and removal of the side chain protecting groups was performed with TFA/water 19/1 (v/v) for 2.5 h. For cysteine -containing peptides triethylsilane was added to the cleavage cocktail. Peptides were isolated and purified by repeated ether precipitations, and were 60-75% pure according to analysis by RP-HPLC. Analysis of products by MALDI-tof mass spectrometry using internal mass calibration showed the expected molecular masses.

Microorganisms and culture conditions

Bacillus subtilis ATCC6633, Escherichia coli ML35 (Lehrer, RI et al. 1989.

Interaction of human defensins with Escherichia coli: mechanism of bactericidal activity. Journal of Clinical Investigation 84:553-561.), Staphylococcus aureus 42D (Zaat, SAJ et al. 1994. Initial characterization of antibacterial proteins from thrombin- stimulated platelets involved in clearance of Streptococcus sanguis from cardiac vegetations in experimental endocarditis, p. 473-475. In A. Totolian (ed.), Pathogenic streptococci: present and future. Lancer Publication, St. Petersburg.), S. aureus strain JAR060131 ( Campoccia et al. (Int J Artif Organs. 2008

Sep;31(9):841-7), Staphylococcus epidermidis RP62a, Pseudomonas aeruginosa, Eschericia coli ESBL and the fungi Candida albicans, Cryptococcus neoformans and Aspergillus niger (clinical isolates) were used as standard test strains. Selected peptides were also tested for activity against Staphylococcus epidermidis RP62a (ATCC35984) (Christensen, GD et al. 1982. Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infection and Immunity 37: 318- 326.), Pseudomonas aeruginosa PAOl (ATCC15692), Pseudomonas aeruginosa LUH 15100, a clinical isolate of extended-spectrum beta-lactamase producing (ESBL) E. coli, Staphylococcus aureus LUH 15095, Staphylococcus aureus

LUH15096, MRSA LUH 14616, MRSA LUH 15094, Enterococcus faecium

LUH 10330, Klebsiella pneumoniae LUH8995 and Acinetobacter baumannii

RUH875. Bacteria were maintained on blood agar plates, and C. neoformans and A. niger on Isosensitest agar plates (Oxoid, Unipath, Basingstoke, UK).

For antimicrobial assays, bacteria were cultured overnight in Tryptic Soy Broth (TSB, Difco, MI, USA) at 37°C. These cultures were diluted 50-fold in fresh TSB and grown to log-phase in 2-3 h. C. neoformans was cultured at 30°C for 48 h in 0.7% [w/v] yeast nitrogen base (YNB; Difco) supplemented with 0.15% [w/v] L- asparagine (Merck, Darmstadt, Germany) and 1% [w/v] glucose (Merck).

Liquid microbicidal assay

Microbicidal activity of peptides in 0.06% [w/v] TSB was quantified in liquid assays as follows. Bacterial or fungal cells were washed in 10 mM phosphate buffer pH 7.0, supplemented with 0.06% [w/v] TSB, and diluted to 1-2 x 10⁵ cfu/ml in this solution. In initial screening peptides were tested at a final concentration of 60 μΜ. For analysis of LC99.9 solutions of 120 μΜ and serial 2-fold dilutions thereof were tested. Aliquots of 5 μΐ peptide solution in 0.01% acetic acid were transferred to a low protein-binding polypropylene microtiter plate (Costar, Cambridge, USA) and to each well 45 μΐ of bacterial or fungal suspension containing 0.5- lxlO⁴ cfu, was added. The plate was incubated on a rotary shaker (150 rpm) at 37°C (bacteria) or 30°C (C. neoformans) for 2 h. Under these conditions the numbers of test microorganisms did not increase or decrease significantly in control incubations without peptide. Duplicate 10 μΐ aliquots of each incubation were spotted on blood agar plates which had been pre-dried for 1 h at 37°C. Plates were incubated at 37°C (bacteria) or 30°C (C. neoformans) and inspected the next day (bacteria) or after 48 h (C. neoformans). The LC99.9 is defined as the concentration of peptide which had killed > 99.9% of the inoculum in each of the replicate incubations over the period of 2 hours. LC99.9in duplicate or triplicate experiments never differed by more than 1 dilution step.

Microbicidal activity of peptides in PBS or in the presence of 50% plasma was quantified as follows. Peptides were incubated with lxlO⁶ CFU/ml of a mid- logarithmic suspension of S. aureus strain JAR060131 in phosphate buffered saline (PBS; pH 7.4), without or with addition of a pooled human plasma at a final concentration of 50%. Antimicrobial activity is expressed as the 99.9% lethal concentration (LC99.9), i.e., the lowest peptide concentration at which 99.9% of bacteria were killed after 2 hours of incubation at 37°C under shaking conditions. Microbicidal activity of peptide TC19 coupled to cell penetrating peptides in PBS and in the presence of 50% plasma was determined in the same way. The following cell penetrating peptide domains were coupled to peptide TC19:

ARKKAAKAARKKAAKAGG, PLIYLRLLRGQFAGG, PRRPRRPRRGG,

RQIKIWFQNRRMKWKKGG or RWRRWWRRWGG.

In a further experiment, selected peptides were incubated for 0, 1 or 2, or up to 22 hours in 50% plasma, and subsequently incubated for 2 hours with S. aureus to determine the LC99.9 value.

Determination of activity against Aspergillus niger

The effect of the peptide TC19 on fungal growth on Aspergillus niger was assessed by incubation of peptides with 7.5xl0⁴ spores/ml of A. niger PagsA-lux in PBS without or with addition of pooled human plasma at a final concentration of 25%. As positive control, spores were treated with the antifungal caspofungin

(Cancidas). Absorbance was measured over time and at 16 hours, fungal growth was visualized using light microscopy.

Determination of antibiofilm activity

Peptide TC19 was incubated with lxlO⁸ CFU/ml of S. aureus JAR060131 in biofilm-adjusted BM2 in 96-wells polypropylene flat bottom plates. As untreated control, S. aureus was incubated with BM2 medium alone. As background control, no bacteria were added to the BM2 medium. After 24 hours incubation at 37°C, planktonic bacteria were removed by four washes with PBS and biofilms were stained with crystal violet. After solubilization with ethanol, the optical density at 590 nm was determined as a measure of biofilm mass. Biofilm formation is shown in OD 595 nm values relative to the background control and as percentages relative to the BM2 medium (0) treated sample. Antibiofilm activity is expressed as the 50% inhibitory concentration (IC50), i.e., the lowest peptide concentration that resulted in >50% reduction of biofilm mass.

In a further experiment, S. aureus JAR060131 was cultured to mid-logarithmic phase at 37°C under vigorous shaking and then washed once with PBS. This bacterial suspension was diluted in biofilm-adjusted BM2 medium (62 mM

potassium phosphate buffer (pH 7), 7 mM (NH4)2S04, 2 mM MgS04, 10 μΜ FeS04, 0.4% (wt/v) glucose and 0.5% (wt/v) casamino acids) to 2x108 CFU/ml, as calculated from the absorbance of the suspension at 600 nm. In wells of a 96-wells polypropylene flat bottom plate, 50 μΐ of the bacterial suspension were added to 50 μΐ of peptide solution (with final concentrations ranging from 1.6 μΜ - 12.8 μΜ) in BM2 medium. As untreated control, bacteria were exposed to BM2 medium without peptides. After 24 h incubation at 37°C, planktonic bacteria were removed by four washes with PBS and biofilms were stained with 1% crystal violet (Sigma- Aldrich, Zwijndrecht, the Netherlands). After four washes with water and solubilization of the remaining crystal violet with 96% ethanol, the optical density at 595 nm was determined as a measure of biofilm mass. Antibiofilm activity is expressed as the 50% inhibitory concentration (IC50), i.e. the lowest peptide concentration that resulted in >50% reduction of biofilm mass. To assess the antibiofilm activity of the peptides in the presence of plasma, 96- wells polypropylene plates were coated with plasma by overnight incubation with 20% (v/v) human plasma (Sanquin) at 4°C as described by Walker et al.( Front Cell Infect. Microbiol. 2, 39 (2012)) Wells were washed twice with sterile water and then inoculated with S. aureus and peptides as described above.

Immunomodulatory activity

LPS -neutralizing activity

Twohundredfifty ng/ml of LPS (E. coli) was pre-incubated with 0.002-156.25 μΜ of TC19 in PBS for 30 min at 37°C. Next, the LPS-peptide mixture was added 1:200 to blood from a healthy volunteer, yielding final concentrations of 1.25 ng/ml LPS and of 0.01-781.3 nM TC19. After 18-20 h of incubation at 37°C in a 5% CO2 atmosphere, the levels of LPS-induced IL- 12p40 in the supernatants were determined using ELISA.

S. aureus neutralizing activity

A suspension of 5xl0e8 CFU/ml of UV-killed S. aureus JAR was pre-incubated with 9.8-625 μΜ of TC19 in PBS for 30 min at 37C. Next, the S. aureus -peptide mixture was added 1:200 to blood from a healthy volunteer, yielding final concentrations of 2.5xl0e6 CFU/ml S. aureus and of 0.05-3.125 μΜ TC19. After 18-20 h of incubation at 37°C in a 5% CO2 atmosphere, the levels of LPS-induced IL-8 in the

supernatants were determined using ELISA.

Hemolytic activity

A 0.5% erythrocyte suspension was incubated with 0.2-204.8 μΜ of TC19, PBS (background control) or 2.5% Triton-X (100% lysis control) in PBS without or with 50% human plasma. After 1 h of incubation at 37°C, the optical density at 415 nm of the supernatants was determined. The percentage hemolysis was calculated relative to the 100% lysis control.

Results

Identification of microbicidal peptides To identify peptides derived from TC with antimicrobial activity we synthesized a series of 15 overlapping pentadecamers shifted by five residues, covering the entire sequence of CTAP-III encompassing the sequences of TC-1 and TC-2. Peptides were named after their first amino acid and the position of this amino acid in TCI (Figure 1).

Microbicidal activity of the peptides was tested at a 60 μΜ concentration against B. subtilis, S. aureus, E. coli and C. neoformans. Peptides Al from the N-terminal region and D51 from the C-terminal region were active against B. subtilis, E. coli and C. neoformans, causing a 2-3 log decrease in viable counts (Figure 2). S. aureus was not killed by any of the peptides derived from the C-terminal part of TC.

To further define N- and C-terminal microbicidal domains, two additional sets of peptides with a one-residue offset between neighbouring peptides were tested. One set consisted of 6 pentadecamers around peptide A16 in the N-terminal region, the other set of 4 pentadecamers between peptides D51 and K56 in the C-terminal region (Table 1). Of this new set of peptides and of peptides Al, M6, D51 and K56 the LC99.9 for B. subtilis, S. aureus, and E. coli, and the MFC for C. neoformans were determined (Table 1).

Of the N-terminal region peptides, Al was cidal for all organisms at 15-30 μΜ except for S. aureus, which survived even at 120 μΜ. Peptide L3, a peptide shifted two positions to the C-terminus relative to A16, was bactericidal for S. aureus at 30 μΜ, and showed at least a 4-fold increase in activity against the other organisms compared to Al (Table 1). R4, the peptide shifted one more position to the C- terminus was slightly less active than L3, and the next peptide, C20, was active against B. subtilis and C. neoformans at a concentration of 60 μΜ. Apparently, of all tested pentadecamers of the N-terminal region of TCs, peptide L3 optimally encompasses the domain responsible for microbicidal activity.

The activity of the C-terminal region peptides against the test organisms varied (Table 1). Although less than the N-terminal peptide L3, D51 and A52 were active. The C-terminally located peptides P53 and 155 were inactive against E. coli and S. aureus at concentrations of up to 120 μΜ, but were active at high concentration against B. subtilis and C. neoformans (60- 120 μΜ). R69 was fungicidal for C.

neoformans (30 μΜ), but was active against B. subtilis only at high concentration (60-120 μΜ). E. coli and S. aureus were hardly susceptible to any of the C-terminal peptides. Only at 120 μΜ, the highest concentration tested, D51 and A52 killed E. coli and S. aureus, respectively (Table 1).

The antimicrobial peptides identified were all localized within the part of the CTAP-III sequence comprising TC- 1 and TC-2. The most active peptides were derived from the N-terminal part of TCs (e.g. peptides A1-C5), rather than from the C-terminal region.

Variants of peptide L3

Variant peptides of L3 were synthesized to identify amino acids essential for microbicidal activity, and to possibly identify peptides with increased activity.

A derivative of peptide L3 was synthesized in which both cysteines were replaced by serines (L3[SS]). Compared to L3, this peptide had severely reduced activity against B. subtilis and C. neoformans, and was not active at all against E. coli and S. aureus (Table 2). To test whether this difference could be due to differences in dimerization by formation of disulphide bridges, L3 and L3[SS] were analyzed by MALDI-tof mass spectrometry. Since no dimers of non-modified L3 were observed (not shown), it is unlikely that the difference in activity between L3 and L3[SS] was due to dimerization of L3 by disulfide crosslinking, which is not possible in L3[SS].

As net positive charge is thought to be an essential characteristic of antimicrobial peptides, allowing their association with the negatively charged microbial surface and subsequent insertion into target membranes, 3 peptides were synthesized with reduced positive charge, by replacing the basic residues of L3 by the neutral residue alanine (R4A, K9A and K17A, Table 2). Conversely, 12 analogues of L3 were synthesized in which the basic residue lysine did replace the respective neutral amino acid ("K-scan", Table 2). LC99.9 of these peptides for B. subtilis, E. coli and S. aureus, and MFCs for C. neoformans were determined (Table 2).

In all cases the substitution of a charged residue by an alanine (R4A, K9A and K17A) resulted in peptides that have microbicidal activity, although with slightly reduced the activity compared to that of the parent peptide L3 (Table 2). The 3 basic residues in L3 thus are important for microbicidal activity against all organisms tested.

Replacement of any of the neutral residues by the positively charged lysine resulted in increase of activity against S. aureus. Apparently, the presence of positively charged residues, irrespective of their position in the peptide, is of major importance for staphylocidal activity. Against B. subtilis, E. coli and C.

neoformans, lysine substitutions at positions 12 through 16 (numbering of TC-1; peptides S12K - P16K) increased microbicidal activity, but substitution at positions 3 through 11 (peptides L3K - T11K) generally decreased the activity relative to L3 (Table 2) despite the increase of net positive charge.

Multiple variants of L3 were synthesized in which the two central threonines are substituted by tryptophan, tyrosine or phenylalanine and the isoleucine at position 12 is substituted by lysine (see Table 3). All of these variants have antimicrobial activity against S. aureus in PBS (LC99.9 of <60 μΜ), against S. epidermidis in PBS, against P. aeruginosa in PBS and against E. coli in PBS (tables 3, 5, 6, 8). Peptides TC19, TC37, TC43, TC57, TC63, TC69, TC70, TC75 and TC79 show particularly high antimicrobial activity. Moreover, peptides TC19, TC69 and TC75 retain high activity in the presence of 50% pooled human plasma (Table 3). The activity of those peptides was not reduced after 2 hours pre -incubation compared to the assay where bacteria were immediately exposed to peptides in 50% plasma (Table 4). Peptides TC19, TC82, TC84, TC85 and TC94 also retain high activity in the presence of 50% pooled human plasma for prolonged pre-incubation periods of up to 22 hours (Table 10). TC82, TC85 and TC94 show a pattern of plasma stability similar to that of TC19, where TC82 and TC85 have slightly better bactericidal activity than TC19, while TC94 has slightly lower bactericidal activity. TC84 however has superior activity, with very low LC99.9 values for all pre-incubation periods. After plating of the LC99.9 assay, only a few CFUs were present for all pre-incubation periods (so, survival is very close to the LC99.9 cut-off). This indicates that the LC99.9 is most likely 8 μΜ, and that the activity of this peptide is not at all reduced by pre-incubation in plasma up to at least 22 h.

Substitution of amino acids residue by an alanine in peptide TC19 resulted in peptides that have microbicidal activity (Table 7). For activity in PBS, about half of the residues of TC19 are essential. For high antimicrobial activity in the presence of plasma, about half of the residues are essential. Mainly in the N-terminal region some Ala-substitutions can even improve the activity as compared to TC19, see peptides TC83, TC85 and TC86, which have Ala-substitutions at amino acid position 2, 4 and 5, respectively.

Substitutions of amino acids residues by their corresponding D-amino acid in peptide TC19 resulted in peptides with high microbicidal activity against E. coli of all peptides while some of these peptides also showed activity against C. albicans (Table 8).

When peptide TC19 is coupled to cell penetrating peptides (CPP), antimicrobial activity in PBS is fully maintained. In addition, these CPP-coupled TC19 peptides show antimicrobial activity in the presence of plasma as well, although not always to the same degree (table 14).

Antibacterial activity of TC19

TC19 significantly inhibited growth of Staphylococcus aureus JAR060131,

Staphylococcus aureus LUH15095, Staphylococcus aureus LUH15096, MRSA LUH14616, MRSA LUH15094, Staphylococcus epidermidis RP62a, Enterococcus faecium LUH 10330, Klebsiella pneumoniae LUH8995, Acinetobacter baumannii RUH875, Pseudomonas aeruginosa PAOl and Pseudomonas aeruginosa LUH15100 in PBS and Staphylococcus aureus JAR060131, Staphylococcus aureus LUH15095, Staphylococcus aureus LUH15096, MRSA LUH14616, MRSA LUH15094,

Staphylococcus epidermidis RP62a, Enterococcus faecium LUH 10330, Klebsiella pneumoniae LUH8995 and Acinetobacter baumannii RUH875 in the presence of 50% plasma (Table 9).

Further, at >lx LC99.9 of TC19 effectively killed S. aureus JAR within 30 min (Figure 6).

Antifungal activity of TC19

TC19 significantly inhibited A. niger growth at concentrations of 3.2 μΜ in PBS (Figure 3). As plasma influenced the optical density values, the antifungal activity of TC19 in the presence of plasma was assessed based on the light micrographs only. In the presence of 25% plasma, fungal growth was inhibited by 12.8 μΜ of TC19 (Figure 4). Further, TC19 and several variants, including variants containing one or more D- amino acids inhibited C. albicans growth (Table 8).

Antibiofilm activity of TC19

TC19 effectively reduces in vitro biofilm formation by S. aureus JAR (Figure 5). TC19 showed >50% inhibition (IC50) of biofilm formation at 12.8 μΜ in BM2 medium and at 6.4 μΜ (range 3.2-6.4 μΜ) in plasma (Figure 5). The maximal biofilm inhibition measured in BM2 medium was approximately 75% by TC19 at a concentration of 25.6 μΜ. Inhibitory Concentration 50 (IC50) is defined as lowest peptide concentration that resulted in at least 50% reduction in biofilm mass.

Immunomodulatory activity

Concentrations of 1.25-6.25 μΜ TC19 inhibit >90% of the LPS-induced IL-12P40 production in whole blood (Table 11), and concentrations of 0,391 - 0,781 μΜ TC19 inhibit >90% of the S. aureus -induced IL-8 production in whole blood (Table 12).

Hemolytic activity

In PBS, up to 51.2 μΜ of TC19 caused less than 20% of erythrocyte lysis. In plasma, TC19 caused less than 5% erythrocyte lysis at any of the concentrations tested up to 204.8 μΜ. Table 1. Sequences of thrombocidin-derived peptides, their overall charge at neutral pH, and their microbicidal activity (μΜ) against the microorganisms studied as expressed by the LC99.9.

Peptide Sequence Charge B. E. coli S. aureus C. neoformans subtilis

L-2 LYAELRCMCIKTTSG + 1 >120 >120 >120 >120

Y- l YAELRCMCIKTTSGI + 1 >120 >120 >120 >120

Al AELR CMCIKTTSGIH + 1 15 30 >120 15

E2 ELRCMCIKTTSGIHP + 1 30 60 >120 7.5

L3 LRCMCIKTTSGIHPK +3 3.8 7.5 30 1.9

R4 RCMCIKTTSGIHPKN +3 15 7.5 >120 3.8

C5 CMCIKTTSGIHPKNI +2 60 >120 >120 60

M6 MCIKTTSGIHPKNIQ +2 >120 >120 >120 >120

D51 DAPRIKKIVQKKLAG +4 30 120 >120 30

A52 APRIKKIVQKKLAGD +4 30 >120 120 30

P53 PRIKIvIVQKKLAGDE +3 120 >120 >120 60- 120

R54 RIKKIVQKKLAGDES +3 60- 120 >120 >120 30

155 IKKIVQKKLAGDESA +2 >120 >120 >120 60- 120

K56 KKIVQKKLAGDESAD + 1 >120 >120 >120 >120

Table 2. Sequences of peptides derived from peptide L3 by alanine and ly

modifications (underlined), their overall charge at neutral pH, and their

microbicidal activity (μΜ) against the microorganisms studied as expressed by the LC99.9. Modifications are underlined.

LC99.9 (uM) for

Peptide Sequence Charge B. E. coli S. C.

subtilis aureus neoformans

L3 LRCMCIKTTSGIHPK +3 3.8 7.5 30 1.9

R4A LACMCIKTTSGIHPK +2 15 30-60 120 30

K9A LRCMCIATTSGIHPK +2 15 120 60 15

K17A LRCMCIKTTSGIHPA +2 120 >120 >120 30

L3K KRCMCIKTTSGIHPK +4 3.8 >120 15 7.5

C5K LRKMCIKTTSGIHPK +4 15 >120 15 3.8

M6K LRCKCIKTTSGIHPK +4 7.5 >120 7.5 3.8

C7K LRCMKIKTTSGIHPK +4 15 >120 15 7.5

I8K LRCMCKKTTSGIHPK +4 15 >120 7.5 30-60

T10K LRCMCIKKTSGIHPK +4 7.5 >120 3.8 3.8

T11K LRCMCIKTKSGIHPK +4 3.8 120 3.8 3.8

S 12K LRCMCIKTTKGIHPK +4 1.9 7.5 1.9 3.8

G13K LRCMCIKTTSKIHPK +4 3.8 7.5 1.9 1.9

I14K LRCMCIKTTSGKHPK +4 0.9 3.8 3.8 0.9

H15K LRCMCIKTTSGIKPK +4 1.9 3.8 30 1.9

P16K LRCMCIKTTSGIHKK +4 1.9 15 3.8 1.9

L3[SS] LRSMSIKTTSGIHPK +3 60- 120 >120 >120 60- 120

Table 3. Sequences of peptides derived from peptide L3, and their microbicidal activity against S. aureus in PBS without or with addition of a pooled human plasma at a final concentration of 50% expressed by the 99.9% lethal concentration (LC99.9), i.e., the lowest peptide concentration at which 99.9% of bacteria were killed after 2 hours of incubation at 37°C under shaking conditions. Amino acid changes are depicted in bold characters. Ac = acetyl group, B = amide group

TC 55 WWLRCMCIKWWWSGKHPKB <60 >60

TC 56 WWRCMCIKWWWSGKHPKB <60 >60

TC 57 LRCMCIKFFSGKHPKB 8 >60

TC 58 LRCMCIKFFFSGKHPKB <60 >60

TC 59 FFLRCMCIKWWSGKHPKB <60 >60

TC 60 FFLRCMCIKWWWSGKHPKB <60 >60

TC 61 FFLRCMCIKFFSGKHPKB <60 >60

TC 62 FFLRCMCIKFFFSGKHPKB <60 >60

TC 63 LRCMCIKYYSGKHPKB 15 >60

TC 64 LRCMCIKYYYSGKHPKB <60 >60

TC 65 YYLRCMCIKWWSGKHPKB <60 >60

TC 66 YYLRCMCIKWWWSGKHPKB <60 >60

TC 67 YYLRCMCIKYYSGKHPKB <60 >60

TC 68 YYLRCMCIKYYYSGKHPKB <60 >60

TC 69 Ac-LRCMCIKWWSGKHPKB 8 15-60

TC 70 Ac-WWLRCMCIKWWSGKHPKB 4-8 >60

TC 71 Ac-FFLRCMCIKFFSGKHPKB <60 >60

TC 72 Ac- LRCMCIKWWWSGKHPKB <60 >60

TC 73 Ac-WWLRCMCIKWWWSGKHPKB <60 >60

TC 74 Ac-FFLRCMCIKFFFSGKHPKB <60 >60

TC 75 LRCMCIKWWSGKKPKB 4-8 15-60

TC 76 LKCMCIKWWSGKHPKB <60 >60

TC 77 LRCMCIRWWSGRHPRB <60 >60

TC 78 LRCMCIKWWWSGKKPKB <60 >60

TC 79 LRCMCIKWWWSGKHPKB <60 >60

TC 80 LRCMCIRWWWSGRHPRB <60 >60

Table 4. LC99.9 (μΜ) of peptides TC19, TC43, TC57, TC63, TC69, TC70 and TC75, against S. aureus strain JAR060131 after pre-incubation in 50% plasma.

pre-incubation time

Peptide nr. O h l h 2 h

19 30 60 30

43 120-240 120 - >240 >240

57 >240 240 240

63 240 240 240

69 30-60 60 30-60

70 >240 >240 >240

75 8- 15 30 15 Table 5. LC99.9 (μΜ) of TC8 (=L3), TC18, TC19 and TC23 against S. epidermidis

RP62a in PBS

Table 6. LC99.9 (μΜ) of TC19, TC37, TC39, TC43, TC57, TC63, TC69, TC70 and

TC75 against P. aeruginosa in PBS

Table 7. LC99.9 (μΜ) of alanine -scan peptides of TC19 against S. aureus strain JAR060131. TC81 is identical to TC19, but synthesized in the same batch as all alanine-scan peptides. B = amide group

PBS 50%

plasma

TC81 8-15

LRCMCIKWWSGKHPKB 8- 15

(=TC19)

TC82 ARCMCIKWWSGKHPKB 15 30

TC83 LACMCIKWWSGKHPKB 4-30 4

TC84 LRAMCIKWWSGKHPKB 15-30 >120

TC85 LRCACIKWWSGKHPKB 4-8 4-8

TC86 LRCMAIKWWSGKHPKB 30-60 8

TC87 LRCMCAKWWSGKHPKB >120 >120

TC88 LRCMCIAWWSGKHPKB 8-15 >120

TC89 LRCMCIKAWSGKHPKB >120 >120

TC90 LRCMCIKWASGKHPKB 60 >120

TC91 LRCMCIKWWAGKHPKB 8-30 >120

TC92 LRCMCIKWWSAKHPKB 8-15 30

TC93 LRCMCIKWWSGAHPKB 15-60 60-120

TC94 LRCMCIKWWSGKAPKB 4-15 >120

TC95 LRCMCIKWWSGKHAKB 8-15 30-60

TC96 LRCMCIKWWSGKHPAB 30-120 >120 Table 8. LC99.9 (μΜ) values of TC- derived peptides against C. albicans or E. coli ESBL in PBS. Ac: acetyl; Z: aminobutyric acid (Abu); X = diaminobutyric acid

(DABA); lower case letters: D-amino acids

peptide Sequence C.albicans E.coli

ESBL

TC19 LRCMCIKWWSGKHPKB 30-60 15

TC44 LRWMWIKWWSGKHPKB 30-60 60

TC46 LRCMCWKWWSGWHPKB >60 15

TC51 LR WMWIKWW WS GKH PKB >60 15

TC52 LRCMCWKWWWSGKHPKB 60 15

TC75 LRCMCIKWWSGKKPKB 30 30

TC78 LRCMCIKWWWSGKKPKB 30 15

TC79 LKCMCIKWWWSGKHPKB 60 <8

TC112 1RCMCIKWWSGKHPKB 60 15

TC113 LrCMCIKWWSGKHPKB 60 15

TC117 LRCMCiKWWSGKHPKB >60 15

TC120 LRCMCIKWwSGKHPKB 30 30

TC122 LRCMCIKWWSGkHPKB >60 15

TC123 LRCMCIKWWSGKhPKB >60 15

TC124 LRCMCIKWWSGKHpKB >60 15

TC126 lrcmcikwwsGkhpkB >60 15

TC127 kp hk Gs w wkicmcrlB 15-30 15

TC128 GRCMCIKWWSGKHPKB 30-60 15

TC131 RRCMCIKWWSGKHPKB >60 15

TC133 LRCMCIKWWSGKHPKB >60 15

TC166 XRCMCIKWWSGKHPKB >60 15

Table 9. Antimicrobial activity of TC19 against different bacterial strains

medi an LC99.9 j { range)

PBS 50% piasma

Gram-positive

•MDR isolate

pan-drug fesistani sscSa z

c^,d n ycyd i rj-r esi slan i soSa t

Table 10. LC99.9 values (μΜ) against S. aureus JAR after indicated pre-incubation periods in 100% human plasma.

Table 11. LPS neutralizing activity of TC19.

donor 1 donor 2

nM Mean Stdev Mean Stdev

0 Ϊ 77& 1.^■$¾ 1230 540

0,01 783 222 883 166

0,05 248 99 470 116

0,25 209 4 471 207

1,25 120 .1.70 177 142

6,25 0 0 0 0

31,25 0 0 0 0

156,3 0 0 0 0

781,3 0 0 0 0 Table 12. S. aureus neutralizing activity of TC19.

fL-8 (pg/m{}

donor 1 donor 2

Mean Stdev Mean Stdev

0 10704 1826 2003 S 3878

0,049 7985 198 15119 1789

0,098 8897 233 14825 703

0,195 4479 428 6831 289

0,391 48.1 2 718 7537 512

0,781 5321 115 6564 471

1,563 3406 110 7898 0

3,125 3396 514 13065 468

Table 13. Hemolytic activity of TC19 in PBS and in 50% plasma.

% hemolysis in PBS

μΜ donor 1 donor 2 donor 3 Mean Stdev donor 1 donor 2 donor 3 donor 4 Mean Stdev

0,2 0 2 3 2 2 0 0 2 0 0 1

0,4 0 1 0 0 0 0 2 1 0 1 1

0,8 5 2 1 3 2 0 2 3 0 1 1

1,6 8 1 2 3 4 0 2 3 0 1 2

3,2 6 5 1 4 3 0 1 1 4 1 2

6,4 14 8 4 9 5 1 0 2 0 1 1

12,8 9 9 4 7 3 0 0 0 0 0 0

25,6 10 10 12 11 1 0 0 2 0 0 1

51,2 11 13 14 13 2 0 0 3 0 1 2

102,4 22 25 23 24 1 2 0 3 0 1 2

204,8 44 45 38 42 4 1 0 4 0 1 2

Table 14. Antimicrobial activity of Cell-Penetrating peptide-coupled TC19.

Antimicrobial activity of cell-penetrating (CP) peptide-coupled TC19 against S. aureus JAR060131 in physiological phosphate buffered saline (PBS) and in the presence of 50% human plasma. Results are expressed as lethal concentration (LC) 99.9%, i.e. the lowest peptide concentration in μΜ that resulted in >99.9% killing of bacteria. Amino acid residues of the CPP domains are depicted bold. CP4 is derived from penetratin (Andrea-Anneliese Keller, Franziska Mussbach, Reinhard

Breitling, Peter Hemmerich, Buerk Schaefer, Stefan Lorkowski and Siegmund Reissmann. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells.

Pharmaceuticals 2013, 6, 184-203; doi: 10.3390/ph6020184)

50%

Peptide Amino acid sequence PBS plasma

CP1-TC19 ARKKAAKAARKKAAKAGGLRCMCIKWWSGKHPKB 1-2 8

CP2-TC19 PLIYLRLLRGQFAGGLRCMCIKWWSGKHPKB 2 >120

CP3-TC19 PRRPRRPRRGGLRCMCIKWWSGKHPKB <1 8- 15

CP4-TC19 RQIKIWFQNRRMKWKKGGLRCMCIKWWSGKHPKB <1 8- 15

CP5-TC19 RWRRWWRRWGGLRCMCIKWWSGKHPKB 1 30

Claims

1. A synthetic, isolated or recombinant peptide of 10-25 amino acids comprising at least 10 amino acids of amino acid sequence

LRCMCIKWWSGKHPK, or at least 10 amino acids of a variant of said sequence, said peptide having antimicrobial, antibacterial, antiviral, antifungal, antiparasitic and/or anti-inflammatory activity;

said variant sequence having one or more of the following modifications:

substitution of one of both of C by I or W;

substitution of K at position 12 by I or W;

substitution of L or I by W;

- substitution of one or both of W by F or Y;

substitution of R by K and/or one or more of K by R;

substitution of H by K;

2. Peptide according to claim 1 wherein said variant sequence optionally has one or more of the following modifications:

3. Peptide according to claim 1 or 2 having an N-terminal and/or C-terminal modification, preferably comprising an N-terminal acetyl-, hexanoyl-, decanoyl-, myristoyl-, NH-(CH2-CH2-0)n-CO- or propionyl-residu and/or a C-terminal amide-, NH-(CH2-CH2-0)n-CO-amide-, or one or two amino-hexanoyl groups.

4. A nucleic acid molecule comprising a nucleic acid sequence encoding a peptide according to any one of claims 1-3.

5. A vector comprising the nucleic acid molecule according to claim 4.

6. A pharmaceutical composition comprising the peptide according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, the nucleic acid molecule according to claim 4 and/or the vector according to claim 5 and at least one pharmaceutically acceptable carrier, diluent and/or excipient.

7. Pharmaceutical composition according to claim 6 comprising a controlled release and/or targeted delivery carrier comprising said peptide, whereby said carrier is preferably selected from the group consisting of nanop articles, microp articles, nanocapsules, microcapsules, liposomes, microspheres, hydrogels, polymers, lipid complexes, serum albumin, antibodies, cyclodextrins and dextran.

8. Coating for a biomaterial, medical device and/or implant comprising a peptide according to any one of claims 1-3.

9. Biomaterial, medical device and/or implant provided with a coating according to claim 8.

10. Peptide according to any one of claims 1-3 and/or nucleic acid molecule according to claim 4 for use as a therapeutic or prophylactic agent.

11. Peptide and/or nucleic acid molecule for use according to claim 10 in the treatment of a microbial, bacterial, fungal, viral and/or parasitic infection and/or in the treatment of a condition resulting from a microbial bacterial, fungal, viral and/or parasitic infection.

12. Peptide and/or nucleic acid molecule for use according to claim 11 wherein said treatment is systemic treatment or topical treatment.

13. Peptide and/or nucleic acid molecule for use according to any one of claims 10- 12 in the treatment and/or prevention of biomaterial associated infection.

14. A method for the treatment of a bacterial, fungal, viral and/or parasitic infection in a subject comprising administering to said subject a therapeutically effective amount of a peptide according to any one of claims 1-3, a nucleic acid molecule according to claim 4, or a pharmaceutical composition according to claim 6 or 7.

15. A method for the preparation of a peptide according to any one of claims 1-3 comprising:

providing a nucleic acid molecule comprising a nucleic acid sequence encoding a peptide according to any one of claims 1-3;

transforming a host cell with said nucleic acid molecule;